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[Cancer Research 60, 3389-3393, July 1, 2000]
© 2000 American Association for Cancer Research

Advances in Brief

Liposomal Encapsulation of Topotecan Enhances Anticancer Efficacy in Murine and Human Xenograft Models1

Paul Tardi, Edward Choice, Dana Masin, Thomas Redelmeier, Marcel Bally and Thomas D. Madden2

Inex Pharmaceuticals Corporation, Burnaby, British Columbia, V5J 5J8 Canada [T. D. M.]; Department of Advanced Therapeutics, British Columbia Cancer Agency [P. T., E. C., D. M., M. B.], Vancouver, British Columbia, V5Z 4E3 Canada; and Northern Lipids Inc., Jack Bell Research Centre, Vancouver, British Columbia, V6H 3Z6 Canada [T. R.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Topotecan was encapsulated in sphingomyelin/cholesterol liposomes using an ionophore-generated proton gradient. After i.v. injection, liposomal topotecan was eliminated from the plasma much more slowly than free drug, resulting in a 400-fold increase in plasma area under the curve. Further, high-performance liquid chromatography analysis of plasma samples demonstrated that topotecan was protected from hydrolysis within the liposomal carrier with >80% of the drug remaining as the active, lactone species up to 24 h. The improved pharmacokinetics observed with liposomal topotecan correlated with increased efficacy in both murine and human tumor models. In the L1210 ascitic tumor model, optimal doses of liposomal topotecan resulted in a 60-day survival rate of 60–80%, whereas in a L1210 liver metastasis model, 100% long-term survival (>60 days) was achieved. In contrast, long-term survivors were rarely seen after treatment with free topotecan. Further, in a human breast carcinoma model (MDA 435/LCC6), liposomal topotecan provided greatly improved increase in life span relative to the free drug. These results suggest that liposomal encapsulation can significantly enhance the therapeutic activity of topotecan.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Topotecan is a water-soluble analogue of camptothecin that specifically inhibits the activity of topoisomerase I by stabilizing the topoisomerase I-DNA complex, resulting in lethal DNA strand breaks (1) . As with all camptothecins, however, topotecan undergoes a pH-dependent hydrolysis of the lactone ring to form a relatively inactive carboxylate in aqueous solution. This rapid conversion is also observed in patients after systemic injection of the drug (2) . A potential solution to this problem is to encapsulate topotecan within a liposome. Liposomes accumulate preferentially at tumor sites as a result of their ability to extravasate through "pores" or "defects" in the capillary endothelium. These "pores" appear to be a consequence of the rapid angiogenesis occurring in tumors and are generally not present in normal tissues or organs (3) . Liposomes have previously been used as carriers for anticancer drugs, and they have been shown to reduce side effects, such as anthracycline-induced cardiomyopathy (4) . Liposomes can also provide slow release of an encapsulated drug, resulting in sustained exposure to tumor cells and enhanced efficacy (5, 6, 7) . In the case of camptothecins, liposomes could additionally provide protection against drug hydrolysis. Initial studies confirmed that insertion of camptothecins within a lipid bilayer conferred protection of the lactone species (8) . For therapeutic purposes, however, this approach is limited by the relatively low drug concentrations that can be inserted into a liposomal membrane and the rapid exchange of hydrophobic drugs that occurs from liposomes to other hydrophobic binding sites after i.v. administration (9 , 10) . In the present work, therefore, we have encapsulated topotecan in the aqueous interior of the liposome using an ionophore-induced proton gradient. This approach provides efficient loading at high drug:lipid ratios, avoids drug exchange from the carrier, and provides an acidic environment stabilizing the lactone species. The pharmacokinetic properties and anticancer efficacy of this liposomal formulation are compared to free topotecan in the present work.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Lipids and Chemicals.
Topotecan (Hycamtin, SmithKline Beecham) was purchased from the British Columbia Cancer Agency Pharmacy. Egg sphingomyelin was supplied by Northern Lipids Incorporated (Vancouver, British Columbia, Canada). Cholesterol and the divalent cation ionophore A23187 were obtained from the Sigma Chemical Company (St. Louis, MO). Tritiated [3H]cholesteryl hexadecyl ether (NEN, Boston, MA) was used as a liposome marker. All other chemicals used in these studies were analytical or HPLC3 grade.

Preparation of Liposomes.
Large unilamellar vesicles consisting of egg sphingomyelin and cholesterol (55:45 mole percent) were prepared as previously described by Hope et al. (11) . Briefly, lipids were dissolved in benzene/methanol (95:5 v/v) in the presence of [3H]cholesteryl hexadecyl ether (98,000 dpm/µmol), frozen in liquid nitrogen, and lyophilized under vacuum for 5 h. The dried lipid films were hydrated in 300 mM manganese sulfate, freeze-thawed five times (12) , and size-reduced using high pressure extrusion through two-stacked polycarbonate filters of 80 nm in pore size (Poretics, AMD Manufacturing Inc., Mississauga, Ontario, Canada). The vesicle diameter was typically in the range of 130 ± 20 nm based on quasi-elastic light scattering (Nicomp Particle Sizer Model 270, Santa Barbara, CA). The external buffer of the carrier system was exchanged by dialyzing at 4°C for 48 h against 100 volumes of 300 mM sucrose with buffer changes at 18 and 36 h.

Topotecan Encapsulation.
Topotecan was encapsulated into the liposomes using an ionophore-mediated proton gradient (13) . Drug uptake was performed at 5 mM topotecan and 40 mM lipid in a solution containing 300 mM sucrose, 30 mM EDTA, and 20 mM 2-morpholineethanesulfonic acid (pH 6.0). The divalent cation ionophore A23187 (7 µM final concentration) was first added to the liposomes, and the mixture was incubated at 65°C for 15 min to facilitate A23187 incorporation into the bilayer. Subsequently, topotecan and EDTA were added, and the mixture was incubated at 65°C for 1 h. The extent of encapsulation was determined by passing an aliquot of the sample down a Sephadex G-50 spin column (14) and measuring lipid and topotecan concentration in the eluent. Unencapsulated topotecan and A23187 were removed from the preparation by dialyzing the sample at 4°C for 48 h against 100 volumes of 300 mM sucrose. The efficiency of topotecan loading typically ranged between 90 and 100%.

Pharmacokinetic Studies in BALB/c Mice.
Free topotecan (10 mg/kg) and liposomal topotecan (125 mg/kg lipid, 10 mg/kg topotecan) were injected i.v. into female BALB/c mice, and the plasma elimination of both the lipid carrier and the drug were determined over a 24-h time course. Quantitation of the liposomal carrier in plasma was based on liquid scintillation counting of the nonexchangeable, nonmetabolizable marker [3H]cholesteryl hexadecyl ether (15) . Total topotecan was determined using fluorescence spectroscopy as described in analytical methods.

Tumor Models.
In the L1210 tumor model, female BDF-1 mice weighing 18–20 g were obtained from Charles River Breeding Laboratories. The L1210 cell line was obtained from the National Cancer Institute tumor cell repository and was maintained by serial passage of ascites fluid. Mice in groups of four or five were inoculated on day 0 with 1 x 104 cells i.v. or 1 x 105 cells i.p. In the MDA435/LCC6 tumor model, female SCID/RAG-2 mice weighing 18–20 g were bred by the British Columbia Cancer Agency Joint Animal Facility through a licensing agreement with Taconic (Germantown, NY). The MDA435/LCC6 cell line was kindly provided by Dr. Robert Clarke of the Vincent Lombardi Cancer Center. The cell line has been previously characterized (16) and was maintained by serial passage of ascites fluid. Groups of four mice were inoculated i.p. with 1 x 106 cells on day 0. Treatments in all tumor models were initiated as a single i.v. dose on day 1 or multiple dosing on days 1, 5, and 9. For the treatment groups, drug dosage was adjusted for average body weight for each group. Control animals received injections of sterile saline. Mice were weighed on the day of tumor injection, and weights were recorded daily until the first death within each group. Survival times were recorded as days after tumor cell injection. Because death cannot be used as an end point, mice were evaluated twice daily by trained animal health technicians and sacrificed at the first sign of distress.

Analytical Methods.
Topotecan was quantified using two different methods. In the blood clearance studies, total topotecan was quantified using fluorescence spectroscopy. Briefly, plasma proteins were precipitated by the addition of 200 µl of methanol to 50 µl of plasma, and the sample was centrifuged in an Eppendorf microcentrifuge for 10 min at 3500 rpm. Topotecan was quantified using a Perkin-Elmer LS50 fluorescence spectrometer (Norfolk, CT) set at an excitation wavelength of 380 nm (2.5-nm slit width) and emission wavelength of 518 nm (2.5-nm slit width).

Quantitation of the lactone and carboxylate forms of topotecan was performed by HPLC analysis (17) . Briefly, topotecan was extracted from 50 µl of plasma by diluting the sample in ice-cold methanol (final concentration 80% methanol) to precipitate plasma proteins and solubilize the liposomes. The methanolic solution was stored at -30°C until analysis. These conditions were found to stabilize the lactone species of the drug for several days. Just before HPLC analysis, the samples were diluted with an equal volume of refrigerated water. Standard curves for the two species of the drug were generated by dissolving the drug in either 40% methanol:60% 10 mM citrate buffer (pH 3) for the lactone species or 40% methanol:60% 10 mM borate buffer (pH 9) for the carboxylate species. HPLC analysis used a Waters Novo-pak column (150 x 3.9 mm) with a run time of 15 min at a flow rate of 1 ml/min. A two solvent mobile phase system consisted of mobile phase A (0.6% acetic acid, 1.5% triethylamine in HPLC grade water) and mobile phase B (0.6% acetic acid, 1.5% triethylamine in 47.9% HPLC grade water and 50% acetonitrile). The elution gradient consisted of a mixture of A:B in the following ratios: 78%:22% for minutes 0–4, 50%:50% for minutes 4–8, followed by 78%:22% for minutes 8–15. Under the HPLC conditions outlined above, the carboxylate species of topotecan elutes at 3 min and the lactone species elutes at 7 min.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Liposomal Encapsulation of Topotecan.
Topotecan was encapsulated into vesicles composed of sphingomyelin and cholesterol. These lipid components were selected based on their chemical stability and ability to provide slow, sustained drug leakage (5) . Efficient drug loading was achieved using a manganese ion gradient to drive formation of a pH gradient (13) . In this study, topotecan was loaded into vesicles at a drug:lipid ratio of 1:8 (mole ratio), and trapping efficiencies of 90–100% were achieved over a 1-h incubation at 60–65°C. No drug leakage was seen on storage of liposomal topotecan at 4°C for 3 weeks.

Pharmacokinetics of Liposomal Topotecan.
Pharmacokinetic studies examined plasma elimination rates for both topotecan and the liposomal carrier. After i.v. injection in BALB/c mice, sphingomyelin/cholesterol liposomes show extended blood circulation times (Fig. 1A)Citation . Carrier elimination rates are similar for topotecan-loaded liposomes, mock-loaded liposomes, and empty liposomes, with 15–20% of the injected doses remaining in the circulation at 24 h. This is comparable to circulation lifetimes reported for liposomal systems that contain polyethylene glycol-conjugated lipids (18) . In contrast to free topotecan, which is rapidly eliminated from the plasma, liposomal topotecan shows an extended circulation lifetime, with 23% of the injected dose remaining in the circulation at 4 h (Fig. 1B)Citation . Over 24 h, a 400-fold increase in plasma area under the curve is observed for liposomal topotecan compared to the free drug. From the carrier and drug pharmacokinetic data, it is also possible to calculate the rate of topotecan release from circulating liposomes. As shown in Fig. 1CCitation , sphingomyelin/cholesterol carriers provide sustained drug release over about 24 h.



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  		Fig. 1. Plasma elimination of free and liposomal topotecan. A, mice were injected i.v. with liposomal topotecan ({blacktriangleup}), mock-loaded liposomes (•), or empty liposomes ({blacksquare}). At various times, plasma was collected and liposomal lipid levels were determined ("Materials and Methods"). B, plasma levels of topotecan were determined at various times after injection of free drug (•) or liposomal topotecan ({blacksquare}). C, topotecan release rate from circulating liposomes calculated from data presented in A and B.

 
Topotecan is reversibly hydrolyzed from a closed-ring lactone to an open-ring carboxylate in aqueous solution. The rate of hydrolysis is pH-dependent, with the carboxylate form being favored at physiological pH (19) . Because the lactone form is the active species, we quantified the relative proportions of lactone and carboxylate after the systemic injection of free and liposomal drug. After injection of free topotecan, ~50% of the drug recovered from the plasma is the active lactone form after 5 min (Table 1)Citation . The distribution between lactone and carboxylate changes very little by the 15-min time point. In contrast, liposomal topotecan is protected as the lactone species. Up to 4 h after injection, >95% of the topotecan recovered from plasma was in the active lactone form. At 24 h after injection, the lactone content had decreased to 84%, but the total plasma concentration was still twice the levels detected at 5 min after injection of the free drug. In conclusion, liposomal topotecan was found to selectively maintain the drug in the active lactone form as well as substantially enhance plasma concentrations. To determine if these pharmacokinetic benefits would translate into enhanced efficacy, liposomal topotecan was compared to free drug in L1210 and MDA435/LCC6 tumor models.


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  		Table 1 Analysis of plasma topotecan after injection of free or liposomal drug

 
Antitumor Efficacy of Liposomal Topotecan.
In an L1210 ascites tumor model, mice were inoculated on day 0, and topotecan (free or liposomal) was administered either as a single dose on day 1 or in multiple doses on days 1, 5, and 9. Free topotecan was well tolerated at the doses given, with little change in animal weights after treatment. In the case of the liposomal topotecan, weight loss was observed in the multiple dosing schedule at 8 mg/kg, and one drug-related death was seen in this group. In general, liposomal topotecan caused more weight loss than the equivalent dose of free drug. In subsequent studies, therefore, liposomal topotecan was administered at 50% of the doses used for free drug. In all treatment groups tested, the liposomal form of the drug showed superior activity to free drug. In the single dosing regimen, 10 mg/kg free drug resulted in a median survival time of 10.5 days compared to liposomal topotecan of 25 days. Median survival times could not be determined for the remaining liposomal topotecan groups because of the high incidence of long-term survivors (>60 days). All mice surviving at 60 days were reinjected i.p. with 1 x 105 L1210 cells. The subsequent survival time of the reinjected mice ranged from 8–11 days.

In the second L1210 model, cells were injected i.v., resulting in tumor seeding primarily to the liver. Again, both single and multidose schedules were examined. As observed in the ascites model, liposomal topotecan showed superior activity to the free drug, even at half the free drug dosage (Fig. 2BCitation ; Table 2Citation ). In the groups treated with free drug, no long-term survivors (>60 days) were observed, whereas all mice receiving liposomal topotecan survived beyond 60 days regardless of treatment protocol. At autopsy on day 61, no tumors were found in any of the liposomal topotecan-treated animals.



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  		Fig. 2. Antitumor efficacy of free and liposomal topotecan in L1210 tumor models. Mouse survival after i.p. injection (A) or i.v. injection (B) of L1210 tumor cells on day 0. Single injections of free or liposomal topotecan were administered i.v. on day 1. A, groups shown are control, untreated animals ({blacksquare}); free topotecan, 10 mg/kg ({circ}); free topotecan, 20 mg/kg ({triangleup}); liposomal topotecan, 10 mg/kg (•); and liposomal topotecan, 20 mg/kg ({blacktriangleup}). B, groups shown are control, untreated animals ({blacksquare}); free topotecan, 10 mg/kg ({circ}); free topotecan, 20 mg/kg ({triangleup}); liposomal topotecan, 5 mg/kg (•); and liposomal topotecan, 10 mg/kg ({blacktriangleup}).

 

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  		Table 2 Antitumor efficacy of free and liposomal topotecan

 
The efficacy of liposomal and free topotecan was also examined in a breast cancer tumor model (16) . The human breast cancer cell line MDA435/LCC6 was injected i.p. into SCID mice on day 0, and treatment with drug began as a single dose on day 1 or in multiple doses on days 1, 5, and 9. Free topotecan administered in single or multiple doses had little effect on improving mice survival. The most efficacious dose of free drug was observed at 20 mg/kg (day 1) where a 21% ILS was achieved (Table 2)Citation . In contrast, liposomal topotecan improved on this result >2-fold with a single 5-mg/kg dose (54% ILS) and 5-fold with multiple doses of 2 mg/kg (117% ILS). As observed in the L1210 tumor models, higher doses of liposomal topotecan resulted in animal weight loss and were not pursued.


   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
It has been shown previously that liposomes can reduce the toxicity of anticancer drugs while maintaining or enhancing efficacy (20 , 21) . These benefits derive from the altered pharmacokinetics and biodistribution afforded by the liposomal carrier. In the case of camptothecins, an additional benefit can be achieved by protection of the active lactone species. An earlier study by Burke and Gao (22) showed that large (500-nm diameter) multilamellar vesicles with an acidic interior could stabilize topotecan as the lactone form. Unfortunately, multilamellar vesicles have limited value as drug carriers because of their rapid clearance from the circulation (18) . We therefore examined the application of highly stable sphingomyelin/cholesterol liposomes of ~100 nm in diameter. Defects in the capillary endothelium of tumor vasculature are typically in the size range of 200–600 nm (3) , and therefore, liposomes of 100 nm in diameter can efficiently extravasate and accumulate within the tumor interstitial space (23) . This tumor accumulation is enhanced for systems that display a long circulation lifetime and, as we show here, sphingomyelin/cholesterol liposomes exhibit this property. The blood residency lifetimes we observe are comparable to those of liposomes possessing polyethylene glycol-conjugated lipids (termed Stealth liposomes). This is the result of two main factors. First, the sphingomyelin/cholesterol bilayer is highly rigid and therefore protein binding to the surface is minimized. This reduces the rate of opsonin-induced carrier clearance (24) . Second, liposome clearance rates are influenced by lipid dose because RES clearance mechanisms are partly saturable (18) . The drug:lipid ratio selected for liposomal topotecan provides a lipid dose that avoids rapid carrier clearance by the RES.

An interesting aspect of the present work is that we observe similar plasma elimination rates for topotecan-loaded liposomes and mock-loaded or empty carriers. This result is in contrast to the decreased elimination rates seen for liposomal doxorubicin compared to empty carriers (25) . In the case of liposomal doxorubicin, drug-induced inhibition of RES activity is believed to account for the slower clearance of drug-loaded carriers. This inhibition, or "RES blockade," is believed to reflect cytotoxicity against phagocytic cells responsible for clearance. Because this phenomenon is not observed for liposomal topotecan, it may suggest that topotecan is not inhibiting the nondividing RES cell population. This result supports previously published in vitro studies showing that in the absence of DNA replication, the reversibly stabilized topoisomerase I-DNA complex has minimal effect on cell survival (26) . The absence of nonspecific cell toxicities could prove to be an advantage associated with the use of liposomal topotecan over other liposomal drugs, such as doxorubicin and vincristine.

In addition to increasing topotecan delivery to tumor sites, sphingomyelin/cholesterol liposomes provide sustained drug release over about 24 h. In view of the fact that topotecan activity is cell-cycle-dependent, increasing tumor cell exposure time should increase tumor cell killing dramatically. Previous studies attempting to increase the therapeutic activity of the free drug have met limited success. In a Phase I clinical trial, continuous infusion of free topotecan increased tumor exposure time, but the conversion to the inactive carboxylate form was so rapid that tolerable doses were often too low to provide any antineoplastic effect (27) . This problem is overcome in the present work by using an ionophore-generated ion gradient to create an acidic carrier interior. This protected topotecan as the lactone species for an extended period ensuring that drug released at the tumor site is in the active form.

Initial animal studies evaluated the toxicity and efficacy of liposomal topotecan in the murine L1210 leukemia model. This model has previously been used in several studies that characterized topotecan activity in vivo (2 , 28 , 29) . Higher drug toxicity, evidenced by weight loss, was seen for liposomal topotecan relative to free drug. This result is not surprising in view of the considerable increase in plasma area under the curve (400-fold) observed for the liposomal formulation and the previously reported correlation of plasma topotecan levels with toxicity (2) . Further, a much greater proportion of the drug is preserved as the lactone species when administered in the liposomal carrier. It should be noted that no toxicity (weight loss) was observed in mice administered empty (mock loaded) liposomes at an equivalent lipid dose to that of the highest liposomal topotecan dose. In the L1210 ascites model, liposomal topotecan was much more efficacious than free drug using either a single dose or multidose schedule. Long-term survivors were seen in all liposomal topotecan-treated groups, whereas only one 60-day survivor was achieved in any of the free drug groups. The comparative activities of free and liposomal topotecan were even more pronounced in the L1210 liver metastasis model. All groups treated with liposomal topotecan showed 100% long-term survival with no evidence of tumor at autopsy, even at half the dose of free drug. No long-term survivors were seen in groups treated with free drug. This remarkable improvement in efficacy may be partially related to the fact that liposomes accumulate in the organs of the RES. Because these organs include the liver, the primary site of tumor seeding after i.v. inoculation of L1210 cells, topotecan delivery to the site may be further enhanced over that achieved with the ascitic tumor model. Finally, in a human breast tumor model MDA435/LCC6, improvements in the therapeutic activity of topotecan were also observed for the liposomal formulation. In this model, free drug showed no significant ILS, whereas liposomal topotecan showed significant activity. The combined results from these tumor models indicate that the therapeutic activity of topotecan can be significantly improved by encapsulating the drug within an appropriate liposomal carrier.

Topotecan is a very promising anticancer drug that has shown clinical activity against small cell and non-small cell lung cancer, ovarian cancer, refractory leukemias/myelodysplastic syndromes, and in childhood sarcomas (30) . As for camptothecins in general, conversion of the drug from the active lactone to an inactive carboxylate occurs rapidly in vivo. By encapsulating topotecan within a liposome, we selectively retained the drug in the active lactone form, increased delivery to tumor sites, and provided sustained drug release and hence tumor exposure. These pharmacokinetic changes significantly enhanced the activity of topotecan against both murine and human tumor models. These findings warrant the further development of liposomal topotecan for potential clinical investigation.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Inex Pharmaceuticals Corporation. Additional funding was by the Medical Research Council of Canada (to M. B.) and the National Cancer Institute of Canada (to T. D. M.). Back

2 To whom requests for reprints should be addressed, at Inex Pharmaceuticals Corporation, 100-8900 Glenlyon Parkway, Glenlyon Business Park, Burnaby, British Columbia, Canada V5J 5J8. Back

3 The abbreviations used are: HPLC, high-performance liquid chromatography; ILS, increase in life span; RES, reticuloendothelial system. Back

Received 2/25/00. Accepted 5/ 8/00.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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