A versatile prodrug approach for liposomal core-loading of water-insoluble camptothecin anticancer drugs

Article abstract of DOI:10.1021/ja0256212

We describe a versatile prodrug strategy for loading the liposomal lumen with water-insoluble camptothecins. The procedure involves conversion of an active camptothecin analogue to a 20-OR ω-aminoalkanoanic ester prodrug in which R = CO[CH₂]_nNH₂ and n = 1-3. The basic amino group of the prodrug serves three roles. First, at pH ranges of 3-5, the amine enhances aqueous solubility. Second, it enhances responsiveness to a transmembrane ammonium sulfate gradient across the liposomal bilayer, thereby facilitating active loading of the agent into the liposomal aqueous core. Third, at a physiological pH of 7 or above (the pH to be encountered following drug release at the tumor site), the nucleophilicity of the amine manifests itself and cyclization to the C-21 carbonyl carbon occurs. This cyclization triggers a rapid and convenient nonenzymatic decomposition process that releases active camptothecin. Accordingly, this novel liposomal approach offers a potential system for tumor-targeting prodrugs of many water-insoluble camptothecins, including the highly lipophilic and clinically attractive analogues SN-38, 9-nitrocamptothecin and DB-67. The rate of formation of the active agent at the tumor site can be controlled through the selection of n (the length of the alkyl spacer group). Copyright

Full text of DOI:10.1021/ja0256212

Published on Web 06/07/2002
A Versatile Prodrug Approach for Liposomal Core-Loading of Water-Insoluble
Camptothecin Anticancer Drugs
Xinli Liu, Bert C. Lynn, Junhong Zhang, Lin Song, David Bom, Wu Du, Dennis P. Curran, and
Thomas G. Burke*
Departments of Chemistry and Pharmaceutical Sciences, and The Experimental Therapeutics Program,
Markey Cancer Center, UniVersity of Kentucky, Lexington, Kentucky 40506, Tigen Pharmaceuticals, Inc.,
235 BoliVar Street, Lexington, Kentucky 40508, and Department of Chemistry, UniVersity of Pittsburgh,
Pittsburgh, PennsylVania 15260
Received January 16, 2002
Camptothecin analogues are anticancer agents that act on topo-
isomerase I.1 Topotecan and CPT-11 have received FDA approval,
and several other derivatives are now in pre-clinical and clinical
development. In patients, camptothecins are known to undergo
widespread biodistribution to both tumor and nontumor sites, with
the latter frequently resulting in notable toxicities to healthy tissues
(e.g. bone marrow, gastrointestinal mucosa).2 Attenuation of such
toxicities and tumor-targeting are proven benefits of liposomal core-
loaded drug formulations.3 To date industrial production of core-
loaded liposomal camptothecin formulations has been limited to
the water-soluble camptothecins topotecan, CPT-11, GG-211 (lur-
totecan), and CDK-602. These agents contain basic amines, and
they are loaded into the core of pre-made small unilamellar vesicles
by well-established chemical gradient methods.4 Although liposomal
core-loaded formulations of water-insoluble camptothecins such as
7-ethyl-10-hydroxy-camptothecin (SN-38) and 7-tert-butyldimeth-
ylsilyl-10-hydroxy-camptothecin (DB-67) are of significant clinical
interest,5 these agents lack amino groups and do not load with use
of gradient driven processes. Ester prodrug development is a well-
established approach to derivatizing such agents containing a hy-
droxyl group,6 and here we describe a prodrug approach that allows
water-insoluble camptothecin prodrugs to load into the liposomal
lumen. Novel mechanistic insight into the pH-mediated prodrug
hydrolysis and the influence of ester functionality is provided.
The procedure involves conversion of an active camptothecin
analogue to a 20-OR ω-aminoalkanoanic ester prodrug,7 in which
R ) CO[CH2]nNH2 and n ) 1-3. The basic amino group of the
prodrug serves three roles. First, at pH ranges of 3-5, it enhances
aqueous solubility.8 Second, it enhances responsiveness to a
transmembrane ammonium sulfate gradient across the liposomal
bilayer, thereby facilitating active loading of the agent into the
liposomal aqueous core. Third, at a physiological pH of 7 or above
(the pH to be encountered following drug release at the tumor site),
the nucleophilicity of the amine manifests itself and cyclization to
the C-21 carbonyl carbon occurs. This cyclization triggers a rapid
and convenient nonenzymatic9 decomposition process that releases
active camptothecin. Accordingly, this novel liposomal approach
may allow for the tumor-targeting of water-insoluble camptothecins.
In contrast to 10-OR-CPT-20-OH compounds such as CPT-11,
which are known to exhibit hydrolysis-resistant carbamate bonds
that require enzymatic cleavage,10 reversed-phase high-performance
liquid chromatographic (RP-HPLC) studies of the stabilities of
several 10-OH-CPT-20-OR and CPT-20-OR glycinate ester pro-
drugs (where R ) COCH2NH2) revealed extensive chemical (i.e.
nonenzymatic) decomposition in every case.11 Prodrug decomposi-
tion was observed in phosphate buffered saline (PBS) at pH 7.4,
as well as in human plasma and blood.
The presence of the amine functionality was found to be essential
for the prodrug decomposition. Whereas camptothecin-20(S)-glycin-
ate 1 [1 µM, PBS (pH 7.4), 37 °C] underwent extensive decom-
position (∼90%) within 3 h, its corresponding aliphatic ester ana-
logue camptothecin-20(S)-acetate (differing solely by the replace-
ment of the amino group with a proton) demonstrated excellent
stability in the above fluids for days with negligible evidence of
hydrolysis. Amine-containing esters of camptothecin with longer
alkyl functionalities (R ) COCH2CH2NH2, R ) COCH2CH2CH2-
NH2) also reacted under similar conditions, albeit at a slower rate
(half-lives of 26.4 and 36.7 h, respectively) than their glycinate
counterparts.
To better understand the mechanism of prodrug decomposition,
RP-HPLC coupled with electrospray ionization-tandem mass spec-
trometry (ESI-MS/MS) and 13C, 15N, and 2D NMR experiments
were used to study the decomposition reaction of camptothecin-
20(S)-glycinate. Camptothecin-20(S)-glycinate 1 decomposed to
produce several products12 (see proposed Scheme 1): the closed-
ring lactone form of camptothecin 5, the ring-opened carboxylate
form of camptothecin 6, and two novel decomposition products 3
and 4 generated following the formation of an unusual six-mem-
bered morpholine 2,5-dione ring 2 (or lactam intermediate). The
lactam intermediate arose by intramolecular nucleophilic attack of
the amino group on the lactone E-ring carbonyl carbon of camp-
tothecin and is in fast equilibrium with structures 3 (which we refer
to as the ortho lactone) and 4. Ortho lactone 3 arose by a second
intramolecular reaction within the lactam intermediate and 3 ex-
hibited the same mass as camptothecin-20(S)-glycinate ester but
with a strikingly different fragmentation pattern. 4 arose by a com-
petitive intermolecular reaction to the lactam intermediate and
reacted to release both 5 and 6.13
Likewise, glycinate ester prodrug analogues of other camptoth-
ecins (1 µM, in PBS, pH 7.4), including DB-67 and SN-38,
underwent extensive decomposition (∼90%) within 3 h. HPLC data
for each analogue studied was consistent with the generation of
the novel decomposition products, along with the lactone and
carboxylate forms of the parent drug.12 ESI-MS/MS analysis
indicated that the proposed ortho lactone 3 generated for DB-67-
(20)S-glycinate displayed the same mass as the glycinate ester
prodrug but with a different fragmentation pattern.12 As in the case
for the camptothecin prodrug, the novel degradation products were
generated from the proposed lactam intermediate.
We suggest that the generation of these lactam intermediates is
central to the rapid decomposition of this class of prodrugs. Initiation
of drug release by prodrug hydrolysis at physiological pH is a pre-
* Address correspondence to this author at the University of Kentucky. E-mail:
tgburke@uky.edu.
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J. AM. CHEM. SOC. 2002, 124, 7650-7651
10.1021/ja0256212 CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
Scheme 1 a
a Glycinate ester of CPT (R1 ) H, R2 ) H), of SN-38 (R1 ) CH2CH3,
R2 ) OH), and of DB-67 (R1 ) Si(CH3)2C(CH3)3, R2 ) OH)
Figure 1. (Left panel) Free vs core-loaded CPT-4-aminobutanoate in whole
blood. (Right panel) Free vs core-loaded DB-67-4-aminobutanoate in whole
blood (pH 7.4, 37 °C).
dictable chemical process with less likelihood of interpatient
variability compared to enzyme-mediated drug release. For this
reason we prioritized our subsequent efforts on the liposomal core-
loading of 20-OR prodrugs over the 10-OR prodrug counterparts.
At low pH values in the 3-5 range we found that 20-OR prodrugs
(where R ) CO[CH2]nNH2 and n ) 1-3) displayed markedly
improved stability, thereby facilitating liposomal loading method-
ologies conducted at low pH values. For example, the amounts of
intact camptothecin-20(S)-glycinate (1 µM in PBS, 37 °C) remain-
ing after 2 h at pH values of 7.4, 6.0, 5.0, and 3.0 were 18%, 68%,
95%, and 99%, respectively.
spectrum; and synthesis of new compounds as well as liposome active
loading methods (PDF). This material is available free of charge via
the Internet at http://pubs.acs.org.
References
(1) For a review of the campothecin biochemistry and drug development
see: Liehr, J., Giovanella, B. C., Verschraegen, C. F., Eds. The
Camptothecins: Unfolding Their Anticancer Potential. Ann. N.Y. Acad.
Sci. 2000, 922, 1-363.
(2) (a) Arun, B.; Frenkel, E. P. Expert. Opin. Pharmacother. 1989, 246, 1046-
1048. (b) Ratain M. J. J. Clin. Oncol. 2002, 20, 7-8.
Remote “active” loading of prodrug into premade small unila-
mellar vesicles, with diameters of 100 nm, was carried out by using
transmembrane ammonium sulfate gradients. Prodrug was added
(3) Reviews: (a) Barenholz, Y. Curr. Opin. Colloid Interface Sci. 2001, 6,
66-77. (b) Burke, T.G.; Bom, D. Ann. N.Y. Acad. Sci. 2000, 922, 36-45
(4) (a) Tardi, P.; Choice, E.; Masin, D.; Redelmeier, T.; Bally, M.; Madden,
T. D. Cancer Res. 2000, 60 (13), 3389-93. (b) Sadzuka, Y.; Hirotsu, S.;
Hirota, S. Jpn. J. Cancer Res. 1999, 90 (2), 226-32.
(5) These agents display potent antitopoisomerase I activities and improved
inherent stabilities of the key 20(S)-R-hydroxy-δ-lactone pharmacophore
in blood and tissues.3b
to a liposomal suspension where initially [(NH4)2SO4]CORE
.
[(NH4)2SO4]EXTERNAL; loading of the prodrug occurred as a result
of base exchange (initiated by NH3 gas molecules departing the
liposome).14 Whereas underivatized camptothecin and DB-67
localize predominantly in the bilayer compartment of the liposome,3b
their 20-OR prodrugs, where R ) CO[CH2]nNH2 and n ) 1-3,
loaded with high efficiency (≈60 to 90%) into the core of liposomes
at clinically relevant drug-to-lipid ratios (between 1:4 to 1:20). More
importantly, these core-loaded liposomal formulations of camp-
tothecin 4-aminobutanoate ester and DB-67 4-aminobutanoate ester
exhibited markedly improved stabilities in whole blood relative to
their free forms (Figure 1). Whereas the decomposition of free
prodrug in both cases was extensive, liposomal entrapment
prevented the degradation process from occurring, providing indirect
evidence that the prodrug was effectively retained within the
liposome for periods up to 40 h. These time periods are known to
be sufficient for successful tumor-targeting to be achieved.3
In conclusion, we have demonstrated the utility of a convenient
ester prodrug strategy for the liposomal core-loading of water-in-
soluble camptothecins. Our prodrug approach opens the door for
liposomal loading and tumor-targeting of a much expanded selection
of camptothecins. If premature drug leakage from the particle can
be reduced and tumor-targeting optimized by using agents such as
SN-38 and DB-67 and related 7-silyl-containing camptothecins
(silatecans), their high intrinsic potencies and persistent lactone ring
stabilities suggest they may be of significant value for targeted
delivery.
(6) Denny, W. A. Eur. J. Med. Chem. 2001 Aug, 36 (7-8), 577-95.
(7) (a) Wall, M. E.; Wani, M. C.; Nicholas, A. W.; Manikumar, G.; Tele, C.;
Moore, L.; Truesdale, A.; Leitner, P.; Besterman, J. M. J. Med. Chem.
1993, 36, 2689-2700. (b) See Supporting Information for our novel and
modified semisynthetic procedures of 10-OH-CPT-20-OR and CPT-20-
OR glycinate ester prodrugs, respectively.
(8) (a) Vishnuvajjala, B. R.; Garzon-Aburbeh, A. U.S. Patent 4943579, 1990.
(b) Wadkins, R. M.; Potter, P. M.; Vladu, B.; Marty, J.; Mangold, G.;
Weitman, S.; Manikumar, G.; Wani, M. C.; Wall, M. E.; Van Hoff, D.
D. Cancer Res. 1999, 59, 3424-3428.
(9) Wadkins et al.8b previously employed HPLC and noted that 20(S)-glycinate
esters of camptothecins hydrolyzed in phosphate buffer (pH 7.5) in the
absence of enzymes to release active drug. HPLC peaks were assigned to
the parent drug and the ionized and un-ionized forms of the glycinate
(both lactone and hydroxy acid forms). No discussion of the hydrolysis
mechanism or evidence for the presence of a lactam intermediate and
ortho lactone was presented.8b
(10) Kawato, Y.; Aonuma, M.; Hirota, Y.; Kuga, H.; Sato, K. Cancer Res.
1991, 51 (16), 4187-91.
(11) CPT, SN-38, and DB-67 glycinate esters were studied in this report.
(12) (a) The observed mass values for the protonated molecular (MH+) ions
of CPT glycinate were as follows: 1 (406), 3 (406), 4 (424), 5 (349), 6
(367). (b) CPT glycinate fragmentation pattern [m/z (%)]: 406 (25), 331
(100), 303(30). Ortho lactone fragmentation pattern [m/z (%)]: 406 (100),
388 (40), 303 (20). (c) DB-67 glycinate fragmentation pattern [m/z (%)]:
536 (100), 461 (50), 433 (20). Ortho lactone fragmentation pattern [m/z
(%)]: 536 (100), 518 (10).
(13) A manuscript fully elucidating the hydrolysis mechanism is in preparation.
Evidence for proposed degradation products 3 and 4 includes the
following: (1) glycine carbonyl 13C isotopic labeling NMR experiment
in PBS showing that this carbon exists as a tetrahedral carbon as in 3; (2)
glycine 15N isotopic labeling NMR experiments showing that 3 and 4
have amide bonds; (3) molecular mass of 3 and 4 are consistent with ref
12a; (4) see Supporting Information for the 2D NMR spectrum of the
N-methyl analogue of 3 (this analogue is isolatable due to longer half-
life).
Acknowledgment. This research was supported by NCI grants
(14) (a) Maurer-Spurej, E.; Wong, K. F.; Maurer, N.; Fenske, D. B.; Cullis, P.
R. Biochim. Biophys. Acta 1999, 1416, 1-10. (b) See Supporting
Information for liposomal active loading.
CA63653, CA83362, and CA75751.
Supporting Information Available: Table of structure of camp-
tothecin analogues, experimental procedures for LC/MS; 2D NMR
JA0256212
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