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Paclitaxel-liposomes for intracavitary therapy of intraperitoneal P388 leukemia
CANCER LETTERS Cancer Letters
Paclitaxel-liposomes
107 (1996)
2655272
for intracavitary therapy of intraperitoneal P388 leukemia
Amamath Sharma”, Uma S. Sharma”, Robert M. Straubinger” “Departmenr
of Pharmaceutics,
539 Cooke Hall,
University
at Buffalo,
State University
of New York, Amherst,
NY 14260-1200,
USA
Received 3 June 1996; revision received 20 June 1996; accepted 24 June 1996
Abstract Paclitaxel, a recently approved antineoplastic agent, is cleared slowly from the peritoneal cavity after IP injection, and therefore appears to be promising for intracavitary therapy of malignancies confined to the peritoneal cavity. However the dose-limiting toxicity of Taxol’, the clinical formulation of paclitaxel, was severe abdominal pain, likely caused by the excipients (Cremophor EL@ and ethanol) that are required to overcome low drug solubility. We tested the hypothesis that a liposome-based formulation could modulate paclitaxel toxicity independent of antitumor activity. The dose-dependence of toxicity and antitumor effect of paclitaxel liposomes was evaluated after IP administration against IP P388 leukemia. Liposomal paclitaxel showed antitumor activity similar to that of free paclitaxel (as Taxol@), but was better tolerated by both healthy and tumor-bearing mice.
Keywords:Paclitaxel; Liposomes; P388 Leukemia; Intraperitoneal
1. Introduction
Paclitaxel’, a diterpenoid natural product [l] from the Pacific Yew (Taxus brevifolia), has demonstrated significant antitumor activity when administeredsystemically to patients having advanced and platinumresistant ovarian cancer [Z]. A formulation designated Taxol@has been approved in the US for that indication. A recent phaseI trial to evaluate the toxicity and * Corresponding author. Tel.: +l 716 6452844 243; fax: +l 716 6453693; e-mail: [email protected] ’ In this manuscript, ‘paclitaxel’ is used to denote the anticancer agent ‘taxol’ identified by Wani et al., 1971. Taxol’, the registere.d trademark
of Bristol-Myers
Squib,
Inc.,
denotes
the clinically
approved formulation of paclitaxel that contains ethanol and Cremophor EL@ as excipients. with Taxolml.
‘Free paclitaxel’
is used synonymously
03043835/96/$12.00 0 1996 El sevier Science Ireland PII SO304-3835(96)04380-7
therapy
pharmacology of Taxol@ administered directly into the peritoneal cavity [3] showedextremely slow clearanceof paclitaxel from that compartment, and administration
of a single
IP dose therefore
resulted
in
elevated drug concentrations in the peritoneal cavity for 24-48 h. Because the cytotoxic effects of paclitaxel appearto depend on both the concentration and duration of exposure to cancer cells [4], paclitaxel administered IP may be an effective drug for local chemotherapy of malignancies within the peritoneal cavity, such asovarian cancer. The dose-limiting toxicity of Taxol@ administered IP was severe abdominal
pain at doses greater
than
125 mg/m’ [3]. Abdominal pain may result from paclitaxel itself, from the ethanol/polyethoxylated castor oil (Cremophor EL@) vehicle that is used to solubilize
Ltd. All rights reserved
paclitaxel
for administration,
or from both
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et al. I Cancer
drug and vehicle combined. Recent studies showing that Taxole exerts infusion-site vesicant or toxic effects [5] support such an hypothesis. Several studies have shown that it is possible to eliminate the Cremophor vehicle by encapsulation of paclitaxel in liposomes [6-lo], and previous work in the liposome field has shown that encapsulation can reduce the local irritation and vesicant action of erosive drugs such as doxorubicin, without significant impact on antitumor effect [11,12]. Therefore, there is strong rationale for evaluating the toxicity and antitumor activity of paclitaxel-liposomes after IP administration. 2. Materials
and methods
2.1. Materials Crystalline paclitaxel, Diluent 12 (consisting of Cremophor EL@ (polyethoxylated castor oil) containing 50% absolute ethanol), and paclitaxel dissolved in Diluent 12 (at a concentration of 30 m&5 ml) either were obtained from the National Cancer Institute (Bethesda, MD) or were prepared fresh from crystalline tax01 and pure solvents. Cremophor EL@ was a gift from BASF Corporation (Parsippany, NJ). Phospholipids were purchased from Avanti Polar Lipids (Birmingham, AL) or Princeton Lipids (Princeton, NJ) and stored in chloroform under argon at -70°C. All organic solvents used were reagent grade or better. Female BALB/c and DBA-2 mice were obtained from Harlan Sprague Dawley (Indianapolis, IN). 2.2. Preparation
of paclitaxel-liposomes
Paclitaxel-liposomes were prepared by hydration of a lyophilized powder consisting of paclitaxel and phospholipids, using a method described elsewhere in detail [lo]. Briefly, phosphatidylcholine and phosphatidylethanolamine were dissolved in chloroform at a 9:l molar ratio. Paclitaxel (10 mg/ml in methanol) was added to achieve a final lipid/paclitaxel molar ratio of 4O:l. The solvent was evaporated in a rotary evaporator at 4O”C, and the resulting paclitaxel/lipid film was dissolved in tert-butanol to achieve a lipid concentration of 100 mM. Two to 10 ml aliquots of the butanolic solution were placed in sterile tubes, shell-frozen in liquid nitrogen, and lyophilized for
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24 h. The lyophilized powder was hydrated in a buffer consisting of NaCl (145 mM), N-Tris[hydroxymethyl]-2-aminoethane-sulfonic acid (10 mM), and etbylenediamine tetraacetate (0.1 mM) to produce suspensions of multilamellar vesicles (MLV). Liposomes were analyzed for paclitaxel by reverse-phase HPLC [13] and for phospholipid content by phosphorus assay [14]. 2.3. Cell growth inhibition
experiments
Cells were suspended at a density of 2 x 104/ml in 24-well plates (Costar) and allowed to grow overnight before treatment. Wells in triplicate were exposed to various concentrations of paclitaxel, added either as liposomes or as free drug adsorbed to serum proteins in the absence of organic solvent. Control wells were treated with equivalent volumes of drug-free buffer or media. Cells were counted after 48 h, and the ICss (concentration resulting in 50% growth inhibition) for each concentration-effect curve was calculated graphically. 2.4. Toxicity of paclitaxel-liposomes The maximum tolerated dose (MTD) of each paclitaxel formulation was estimated by IP administration of multiple doses to healthy Balb/c female mice, using 3 animals per group. Each group was injected daily with free paclitaxel in Cremophor/ethanol or with liposomal paclitaxel. The formulations were diluted with saline and administered in a volume of 1 ml. The starting dose was 40 mg/kg, and doses were escalated in 40 m&g increments. Drug-free liposomes or the Cremophor/ethanol vehicle without paclitaxel were used as control treatments. Drug effects were determined by close observation of weight changes; the largest cumulative dose of paclitaxel causing 110% weight loss within 1 week of treatment was defined as the MTD. Animals showing weight loss > 20% were sacrificed, as weight changes of this magnitude often indicate lethal toxicity. 2.5. Animals and tumor model Female DBA-2 mice of 16-20 g were used as hosts for P388, a murine leukemia model used previously in
A. Sharma
et al. I Cancer
paclitaxel development [ 151. The leukemia was initiated by injecting each mouse IP with lo6 viable cells in a volume of 0.5 ml. Mice were randomized into treatment groups, and treatment was started on day 1 after tumor implantation. IP injections of drug were given on 3 consecutive days. The respective vehicles (Cremophor/ethanol or phosphatidylcholine/phosphatidylglycerol liposomes without paclitaxel) were used as control treatments. In the case of Cremophor-containing formulations, the anhydrous Cremophor/ethanol vehicle (Diluent 12) initially was diluted five-fold with saline before use, similar to the clinical procedure for administering Taxole. Drug-containing formulations or vehicles were further diluted with saline and administered in a volume of 1 ml. The dose of vehicle required to administer the highest dose of Taxole tested contained the equivalent of 0.17 ml of the anhydrous Cremophor/ethanol vehicle; that quantity of vehicle was given to control animals. 2.6. End point for assessing anti-leukemia
activity
The percent increase in lifespan (%ILS) was determined as follows: %ILS = [(MDD treated-MDD control)/MDD control] x 1001 where MDD is median day of death. The control groups consisted of tumorbearing mice treated with drug-free liposomes or with Cremophor/ethanol vehicle. The survival data were analyzed for statistical significance using the Cox-Mantel test, as implemented in the program ‘Solid Tumor’ [ 161. All animal experiments were performed in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines and were approved in advance. 3. Results 3.1. Cytostatic activity of paclitaxel liposomes To investigate whether liposomal encapsulation altered paclitaxel potency, the growth inhibitory activity against P388 leukemia was compared in vitro for free paclitaxel and paclitaxel-liposomes. The two formulations were nearly equipotent, with an ICsO of 55 f 10 nM for free paclitaxel (administered without organic solvent) and an I&, of 60 + 5 nM for paclitaxel-liposomes (data not shown). Under
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similar assay conditions [lo], the ICss values for free paclitaxel on various other tumor cell lines ranged from 90 f 10 nM (for Colon-26 murine adenocarcinoma) to 1.5 f 0.7 nM (for A121a, a human ovarian carcinoma). Thus P388 leukemia showed moderate in vitro sensitivity to paclitaxel. 3.2. Toxicity of paclitaxel liposomes. The maximum tolerated dose (MTD) of paclitaxelliposomes was investigated in healthy Balb/c female mice to assist in designing a dosing regimen for antitumor experiments with P388. In previous studies, the single-dose MTD of Taxol’ (free paclitaxel administered in Cremophor/ethanol) was approx. 50 mg/kg by the IP route [8], while paclitaxel-liposomes had a MTD of 2 200 mg/kg [8,9]. The toxicity of the Cremophor vehicle itself contributed to the lower MTD of free paclitaxel. Similar MTD results were obtained here for single injections of drug. Paclitaxel-liposomes showed a cumulative MTD of 240 mg/kg when divided in 3 equal doses and given on consecutive days. In contrast, cumulative doses of only 160 mg/kg free paclitaxel in Cremophor/ethanol were lethal; with the q3d dosing scheme employed here, the MTD for free paclitaxel was approx. 120 mgJkg. Vehicle contributions to the MTD were ruled out by control experiments with vehicle alone; the quantity of drug-free liposomes or Cremophorl ethanol vehicle required to administer cumulative dose of up to 400 mgJkg paclitaxel (in equal injections given on 10 consecutive days) was not toxic upon IP administration (data not shown). 3.3. Antitumor activity The intraperitoneal P388 leukemia model has been used extensively to investigate the antitumor activity of paclitaxel [15], and was selected to evaluate the toxicity and antitumor activity of free and liposomal paclitaxel administered IP. Free paclitaxel dissolved in Cremophor/ethanol (Taxol@) was tested at cumulative doses of 40,60,X0,100 and 120 mg/kg. Paclitaxel incorporated in liposomes was tested at the same doses, and at 200 and 300 mg/kg. The total dose was administered by IP injection in 3 equal fractions on 3 consecutive days, starting on day 1 after tumor implantation. The number of mice in treatment groups
268
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et al. / Cancer
ranged from 5 to 9. Control groups were tumor-bearing animals that were treated with saline or Cremophor/ethanol (Diluent 12) diluted 15 (v/v) with saline. The greatest increase in lifespan mediated by free paclitaxel (as the Taxol’ formulation) was observed with a dose of 80 mg/kg; mice in this group survived approximately 42% longer than did controls (Fig. la; analysis in Table 1). Mice treated with both lower (40 or 60 mg/kg; Fig. la) and higher (100 or 120 mgikg; Fig. lb) doses of free paclitaxel showed a smaller extension of lifespan compared to the group receiving 80 mg/lcg. For mice treated with 120 m&kg free paclitaxel, the lifespan was nearly equivalent to that of untreated control mice (Table 1). Animals in the 100 and 120 m&g groups appeared to have less ascites than did mice treated with 40 or 60 mg/kg (data not shown), suggesting that greater antitumor activity occurred in the high-dose groups, but was accompanied by greater toxicity. The greatest increase in lifespan mediated by paclitaxel-liposomes, approx. 40% greater than untreated controls, was observed in the 80 mg/kg group (Fig. la). At doses of 40 or 60 m&kg liposomal paclitaxel, animals showed approximately 17 or 25% ILS, respectively (Fig. la, Table 1). Paclitaxel-liposomes at doses greater than 80 mg/ kg did not enhance lifespan further; mice in the 100 and 120 mg/kg groups (Fig. lb) showed approximately 25 and 17% ILS, respectively. At the highest doses tested, 200 and 300 mg/kg, mice exhibited signs of toxicity, including reduced survival compared to the vehicle-treated control groups (Fig. lb). The lifespan was only 42% of control for mice treated at 300 mg/kg paclitaxel in liposomes. In order to test the significance of differences in lifespan among various groups, the survival data was subjected to statistical analysis by the Cox-Mantel test, as described in Section 2. Unlike median day of death (MDD) comparisons, Cox-Mantel analysis compared the data from individual animals to calculate the statistical significance of differences among treatment groups. Cox-Mantel analysis showed that the increase in lifespan mediated by both free paclitaxel and paclitaxel liposomes was significant (compared to controls) at the 40, 60, 80 and 100 mg/kg dose levels (P < 0.01). At those dose levels, the free- and lipo-
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107
(1996)
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6
8
10
12
14
16
18
20
22
Number of Days Fig. 1. Antitumor effect of free- or liposomal paclitaxel on P388 leukemia. Itttraperitoneal P388 leukemia was initiated in female DBA mice as described in Section 2. Animals were treated on 3 consecutive days with IP injections of free paclitaxel in Cremophor/ethanoI (Diluent 12) or of paclitaxel-Iiposomes (T-lip), starting on day 1 after tumor implantation. The volume injected was 1 ml. Untreated controls received equivalent volumes of saline or Diluent 12 without paclitaxel. The number of mice per group was 5-9 (n is indicated in Tables 1 and 2). For humane reasons, moribund animals were sacrificed and deaths were recorded as occurring on the following day. (A) Survival of mice treated with paclitaxel in the range of the optimal therapeutic dose; (B) survival of mice treated with paclitaxel in the range of the MTD. Inset: treatment groups; F, free drug; L, liposomal formulation. The number following the letter designation is the cumulative dose of paclitaxel, in mg/kg.
somal paclitaxel formulations showed similar dosedependent effects on lifespan, and the two formulations could not be discriminated statistically
(P > 0.05).
A. Sharm et al. I Cancer Letters 107 (1996) 265-272
269
Table 1 Antitumor activity of free and liposomal paclitaxel administered i.p. against i.p. P388 leukemia Treatment
Dose @wfWW
Total dose
MDD” (range)
% Incre.aaein lifespan (%ILS)
No. of mice per group
Free paclitaxel
13.3 20.0 26.1 33.3 40.0 13.3 20.0 26.7 33.3 40.0 66.7 100.0 0 0
40 60 80 00 120 40 60 80 100 120 200 300 0 0
15 (14-17) 16 (M-21) 17 (15-19) 15 (12-18) 12 (9-20) 14 (13-18) 15 (13-18) 17 (13-20) 15 (11-16) 13.5 (7-18) 9 (7-10) I (7-9) 13 (12-14) 12 (12-13)
25 33 41.7 25 0 16.7 25 40 25 16.7 -25 -41.7 8.3 0
8 9 9 7 5 7 9 9 9 8 5 5 5 5
Liposomal paclitaxel
Diluent 12 Saline
aMDD, median day of death. b%ILS, percent increase in life span.
In order to investigate the effect of doses in the range of the MTD of each formulation, free paclitaxel in Cremophor/ethanol was administered at cumulative doses of 80, 100 and 120 mgjkg, and paclitaxel-liposomes were tested at cumulative doses of 80, 100, 120, 200 and 300 m&kg. For both Cremophor-conmining- and liposomal formulations, the maximum %ILS (approx. 38% greater than controls), was observed with a cumulative dose of 80 mg/kg (Table 2). Treatment with paclitaxel-liposomes at a cumulative dose of 100 mg/kg mediated a modest
ILS (16%), while free paclitaxel at 100 mgfkg resulted in a 28% decrease in median lifespan relative to vehicle-treated controls. Treatment with paclitaxel-liposomes at 120 mgfkg resulted in a median lifespan approx. equal to that of controls, while mice treated with 120 mg./kg free paclitaxel showed a markedly reduced lifespan, 44% shorter than vehicle-treated controls. A dose of 300 m@kg liposomal paclitaxel was required to produce a reduction in lifespan that was roughly similar to the toxic effect observed with 120 mg/kg free paclitaxel (Table 2).
Table 2 Antitumor activity of high-dose paclitaxel administered i.p. against i.p. P388 leukemia Treatment Free paclitaxel Liposomal paclitaxel
Diluent 12 Blank-MLV
26.7 33.3 40.0 26.7 33.3 40.0 66.7 100.0 0.0 0.0
“MDD, median day of death. b%ILS, percent increase in life span.
Total dose
MDD” (range)
80 100 120 80 100 120 200 300 0 0
22 (21-25) 12 (8-25) 7 (7-l 1) 22 (19-23) 19 (13-23) 15 (12-24) 12 (8-24) 7 (7-8) 17 (16-19) 16 (1616)
% Increase in lifespan (%ILS)b
No. of mice per group
38 -28 -56 38 16 -06 -25 -56 cm 00
10 10 5 10 10 5 5 5 5 5
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Cox-Mantel statistical analysis showed that the increase in lifespan mediated by treatment with 80 mg/kg free or liposomal paclitaxel was significant compared to controls (P < O.OOl), but differences between the two formulations could not be discriminated statistically (P > 0.05) at that optimal dose. 4. Discussion Intraperitoneal chemotherapy has been proposed for to improve the management of cancers residing in the peritoneal cavity [ 17,181. From physiological modeling studies and from experimental data, it has been found that the clearance of some anti-neoplastic drugs from the peritoneum is considerably lower than their clearance from plasma [ 171. A blockade of lymphatic drainage may occur in diseases such as ovarian cancer [ 171, and this blockage may reduce further the peritoneal clearance of drugs with particular physical properties, or drugs associated with particulate carriers such as liposomes. For such agents, direct IP administration may result in sustained high drug concentrations in the immediate vicinity of the target site, and enhanced tumor exposure to drug. Paclitaxel appears to have pharmacokinetic properties that make it well-suited for intraperitoneal chemotherapy; after IP administration of Taxole to humans, a high paclitaxel concentration in peritoneal fluids was sustained for long periods [3]. However, severe abdominal pain was dose-limiting, and precluded dose escalation. Drug or vehicle could contribute to the toxicity, and mechanisms likely include direct irritation and chemical peritonitis, analogous to the injection-site vesicant action observed previously [S] . Liposomal encapsulation of erosive drugs such as doxorubicin has been shown to reduce local drug toxicity without a significant impact on antitumor effect [ 11,121, and previous work showed liposomes to be a means to eliminate the Cremophor and ethanol excipients that are used in the Taxol@ formulation [6-lo] of paclitaxel. For drug administered intravenously, liposomal formulations have reduced both the acute, vehicle-dependent toxicity [ 19-221 and the delayed, mechanism-dependent toxicity of paclitaxel itself. The results presented here demonstrate that paclitaxel liposomes administered IP also had lower cumulative toxicity in tumor-bearing mice than did the paclitaxel-
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Cremophor formulation used clinically, and the acute, vehicle-mediated toxicity was eliminated. Although the optimal dose for extending lifespan was similar for both the free- and liposomal paclitaxel formulations, and was achieved at doses significantly lower than the MTD of either formulation, the higher MTD of the liposomal formulation may nonetheless confer clinical advantage. The P388 tumor is moderately sensitive to paclitaxel, and responds to the conventional Cremophor-containing formulation. However, tumors may have significantly greater resistance to the drug, or may acquire resistance through treatment. Previously we compared the antitumor effect of free- and liposomal paclitaxel administered IV against Colon-26 [9], a resistant tumor cited as non-responsive to Taxole Rose, 1992 #154. Growth delay of Colon-26 was not observed with any dose of free paclitaxel (Taxole), including doses having delayed lethality [9]. Growth delay was only observed with liposomal paclitaxel, at doses that would be 100% lethal if given in the Cremophor/ethanol vehicle [9]. Liposomal formulations were well-tolerated, with no drug-mediated lethality observed. Overall, it may be necessary clinically to accept some degree of toxicity in order to exert control of tumor growth, and the liposomal formulation tested here allows the administration of higher paclitaxel doses for a given degree of toxicity. The mechanism by which liposomal encapsulation elevates MTD without altering antitumor effect is not clear. In vitro testing showed that free- and liposomal paclitaxel were equipotent, arguing against a loss of potency in the liposome-based formulation. Alternative hypotheses deserve further exploration, including the possibility that encapsulation in liposomes alters the pharmacokinetics of paclitaxel, and that the pharmacokinetic profiles that mediate toxicity to tumor versus critical tissues are different. The results also suggest that paclitaxel is released from the liposomes to exerts its effect as free drug; the rapidly-growing P388 cells occlude the lymphatic drainage of the peritoneal cavity, and likely restrict the liposomes to the peritoneum. However, the site of dose-limiting toxicity likely is reached through the systemic circulation. If comprehensive pharmacokinetic experiments confirm such an hypothesis, then further alteration of the liposomal vehicle may be possible to enhance the paclitaxel antitumor effect. It may be possible to
A. Sharrm et al. I Cancer Letters IO7 (1996) 265-272
develop liposomes having further-reduced rates of paclitaxel release, thereby enhancing the residence time of drug in the tumor-containing compartment. In conclusion, the experiments presented here suggest that encapsulation of paclitaxel in liposomes may allow the administration of the drug with lower toxicity than observed clinically for equivalent doses of Taxol@, or may allow the administration of higher paclitaxel doses with toxicity equal to that which is accepted currently. Given the interest in treating patients at doses greater than the maximum tolerated dose, which necessitates ‘rescue’ with granulocyte colony-stimulating factor (G-CSF) [23,24], it is possible that the reduction in toxicity achieved by encapsulation in liposomes could reduce or obviate the need for cytokine rescue. The results also underscore the possibility that with some tumors, higher doses of paclitaxel may enhance toxicity without improving overall antitumor efficacy [25]. Finally, a comparison of the liposome-based formulation with the solventbased Taxol@ formulation suggests the intriguing possibility that alterations in paclitaxel formulation may decouple toxicity and antitumor effect. An elucidation of the mechanistic basis for this decoupling could hold considerable benefits to patients, should it be possible to manipulate independently the dose-responses for toxicity and antitumor effect. Acknowledgements Paclitaxel was supplied by the National Cancer Institute, Bethesda, MD. We are indebted to Dr. Matt Suffness (recently deceased), National Cancer Institute, and Dr. Eric Mayhew, now at The Liposome Company (Princeton, NJ) for their enthusiasm, advice and encouragement. This work was supported by grant CA55251 from the National Cancer Institute, National Institutes of Health. References [l]
Wani, M.C., Taylor, H.L., Wall, M.E., Coggon, P. and McPhail, A.T. (1971) Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia, I. Am. Chem. Sot.. 93, 2325-2327. [2] McGuire, W.P., Rowinsky, E.K., Rosenshein, N.B., Grumhine, F.C., Ettinger, D.S., Armstrong, D.K. and Donehower, R.C. (1989) Taxol: a unique antineoplastic
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agent with significant activity in advanced ovarian epithelial neoplasms, Ann. Intern. Med., 111, 273-279. M., Rowinsky, E., Hakes, T., Reichman, B., Jones, [31 Markman, W., Lewis, J.L Jr., Rubin, S., Curtin, J., Barakat, R., Phillips, M., Hurowitz, L., Ahnadrones, L. and Hoskins, W. (1992) Phase I trial of intraperitoneal taxol: a Gynecologic Oncology Group study, J. Clin. Oncol., 10, 148.5-1491. E.K., Donehower, R.C., Jones, R.J. and Tucker, 141 Rowinsky, R.W. (1988) Microtubule changes and cytotoxicity in leukemic cell lines treated with taxol, Cancer Res., 48,4093-4100. C., Burke, T.W., Morris, M., Warner, [51 Bicher, A., Levenbach, D., DeJesus, Y. .and and Gershenson, D.M. (1995) Infusion site soft-tissue injury after paclitaxel administration, Cancer, 76, 116-120. WI Riondel, J., Jacrot, M., Fessi, H., Puisieux, F. and Poiter, P. (1992) Effects of free and liposome-encapsulated tax01 on two brain tumors xenogmfted into nude mice, In Vivo, 6, 23-28. [71 Bartoli, M.H., Boitard, M., Fessi, H., Beriel. H., Devissaguet, J.H., Picot, F. and Puisieux, F. (1990) In vitro and in vivo antitumor activity of free and encapsulated taxol, J. Microencapsulation, 7, 191-197. [sl Straubinger, R.M., Sharma, A., Murray, M. and Mayhew, E. (1993) Novel tax01 formulations: taxol-containing liposomes, J. Natl. Cancer Inst. Monogr., 15, 69-78. R.M. (1993) Anti191 Sharma, A., Mayhew, E. and Straubinger, tumor effect of taxol-containing liposomes in a taxol-resistant murine tumor model, Cancer Res., 53, 5877-5881. 1101Sharma, A. and Straubinger, R.M. (1994) Novel tax01 formulations: preparation and characterization of taxol-containing liposomes, Pharm. Res., 11, 889-896. [ill Forssen, E. and TSk&, Z. (1983) Attenuation of dermal toxicity of adriamycin by liposome encapsulation, Cancer Treat. Rep., 67, 481-484. [121 Mayhew, E., Rustum, Y. and Vale, W. (1984) Inhibition of liver metastases of M5076 tumor by liposome-entrapped adriamycin, Cancer Drug Delivery, 1, 43-58. A., Conway, W. and Straubinger, R. (1994) v31 Sharma, Reversed-phase high performance liquid chromatographic determination of taxol in mouse plasma, J. Chromatogr. B, 655, 315-319. G.R. (1959) Phosphorus assay in column u41 Bartlett, chromatography, J. Biol. Chem., 234, 466-468. 1151Rose, W.C. (1992) Taxol: a review of its preclinical in vivo antitumor activity, Anti-Cancer Drugs, 3, 311-321. [I61 Parsons, J., Bellnier, D., Johnson, P., Oseroff, A., Sharma, A., Bernacki, R. and Greco, W. (1995) Computer hardware/software system for recording and analyzing data from photodynamic therapy and chemotherapy rodent tumor growth, Proc. Am. Assoc. Cancer Res., 36, 609. 1171 Dedrick, R., Myers. C., Bungay, P. and DeVita, V Jr. (1978) Pharmacokinetic rationale for peritoneal drug administration in the treatment of ovarian cancer, Cancer Treat. Rep., 62, l11. chemotherapy in Cl81 Myers, C. (1984) The use of intraperitoneal the treatment of ovarian cancer, Semin. Oncol., 11.275-284. [I91 Lorenz, W., Riemann, H.J., Schmal, A., Schult, H., Lang, S.,
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A. Sharm et al. I Cancer Letters 107 (1996) 265-272 Ohmann, C., Weber, D., Kapp, B., Luben, L. and Doenicke, A. (1977) Histamine release in dogs by Cremophor EL and its derivatives: oxyethylated oleic acid is the most effective constituent, Agents Actions, 7,63-67. Dye, D. and Watkins, J. (1980) Suspected anaphylactic reaction to Cremophor EL, Br. Med. J., 280, 1353. Weiss, R.B., Donehower, R.C., Wiernik, P.H., Ohnuma, T., Gralla, R.J., Trump, D.L., Baker, J.R., VanEcho, D.A., VonHoff, D.D. and Leyland-Jones, B. (1990) Hypersensitivity reactions from taxol, J. Clin. Oncol., 8, 1263-1268. Rowinsky, E.K., Onetto, N., Canetta, R.M. and Arbuck, S.G. (1992) Taxol: the first of the taxanes, an important new class of antitumor agents, Semin. Oncol., 19, 646-662. Rowinsky, E., Chaudhry, V., Forastiere, A., Sartorius, S., Ettinger, D., Grochow, L., Lubejko, B., Comblath, D. and
Donehower, R. (1993) A phase I and pharmacologic study of paclitaxel and cisplatin with granulocyte colony-stimulating factor: neuromuscular toxicity is dose-limiting, J. Clin. Oncol., 11, 2010-2020. [24] Seidman, A., Reichman, B., Crown, J., Yao, T., Heelan, R., Hakes, T., Lebwohl, D., Gilewski, T., Surbone, A., Currie, V., Hudis, C., Klecker, R., Jam&Dow, C., Collins, J., Quinlivan, S., Berkery, R., Toomasi, F., Canetta, R. and Norton, L. (1993) Tax01 plus recombiit human granulocyte-colony stimulating factor as initial and as salvage chemotherapy for metastatic breast cancer: a preliminary report, J. Natl. Cancer Inst. Monographs, 15, 171-176. [25] Markman, M. (1993) Optimal versus maximally tolerated dose in cancer chemotherapy treatment, J. Cancer Res. Oncol., 119, 576-577.
Paclitaxel-liposomes
107 (1996)
2655272
for intracavitary therapy of intraperitoneal P388 leukemia
Amamath Sharma”, Uma S. Sharma”, Robert M. Straubinger” “Departmenr
of Pharmaceutics,
539 Cooke Hall,
University
at Buffalo,
State University
of New York, Amherst,
NY 14260-1200,
USA
Received 3 June 1996; revision received 20 June 1996; accepted 24 June 1996
Abstract Paclitaxel, a recently approved antineoplastic agent, is cleared slowly from the peritoneal cavity after IP injection, and therefore appears to be promising for intracavitary therapy of malignancies confined to the peritoneal cavity. However the dose-limiting toxicity of Taxol’, the clinical formulation of paclitaxel, was severe abdominal pain, likely caused by the excipients (Cremophor EL@ and ethanol) that are required to overcome low drug solubility. We tested the hypothesis that a liposome-based formulation could modulate paclitaxel toxicity independent of antitumor activity. The dose-dependence of toxicity and antitumor effect of paclitaxel liposomes was evaluated after IP administration against IP P388 leukemia. Liposomal paclitaxel showed antitumor activity similar to that of free paclitaxel (as Taxol@), but was better tolerated by both healthy and tumor-bearing mice.
Keywords:Paclitaxel; Liposomes; P388 Leukemia; Intraperitoneal
1. Introduction
Paclitaxel’, a diterpenoid natural product [l] from the Pacific Yew (Taxus brevifolia), has demonstrated significant antitumor activity when administeredsystemically to patients having advanced and platinumresistant ovarian cancer [Z]. A formulation designated Taxol@has been approved in the US for that indication. A recent phaseI trial to evaluate the toxicity and * Corresponding author. Tel.: +l 716 6452844 243; fax: +l 716 6453693; e-mail: [email protected] ’ In this manuscript, ‘paclitaxel’ is used to denote the anticancer agent ‘taxol’ identified by Wani et al., 1971. Taxol’, the registere.d trademark
of Bristol-Myers
Squib,
Inc.,
denotes
the clinically
approved formulation of paclitaxel that contains ethanol and Cremophor EL@ as excipients. with Taxolml.
‘Free paclitaxel’
is used synonymously
03043835/96/$12.00 0 1996 El sevier Science Ireland PII SO304-3835(96)04380-7
therapy
pharmacology of Taxol@ administered directly into the peritoneal cavity [3] showedextremely slow clearanceof paclitaxel from that compartment, and administration
of a single
IP dose therefore
resulted
in
elevated drug concentrations in the peritoneal cavity for 24-48 h. Because the cytotoxic effects of paclitaxel appearto depend on both the concentration and duration of exposure to cancer cells [4], paclitaxel administered IP may be an effective drug for local chemotherapy of malignancies within the peritoneal cavity, such asovarian cancer. The dose-limiting toxicity of Taxol@ administered IP was severe abdominal
pain at doses greater
than
125 mg/m’ [3]. Abdominal pain may result from paclitaxel itself, from the ethanol/polyethoxylated castor oil (Cremophor EL@) vehicle that is used to solubilize
Ltd. All rights reserved
paclitaxel
for administration,
or from both
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drug and vehicle combined. Recent studies showing that Taxole exerts infusion-site vesicant or toxic effects [5] support such an hypothesis. Several studies have shown that it is possible to eliminate the Cremophor vehicle by encapsulation of paclitaxel in liposomes [6-lo], and previous work in the liposome field has shown that encapsulation can reduce the local irritation and vesicant action of erosive drugs such as doxorubicin, without significant impact on antitumor effect [11,12]. Therefore, there is strong rationale for evaluating the toxicity and antitumor activity of paclitaxel-liposomes after IP administration. 2. Materials
and methods
2.1. Materials Crystalline paclitaxel, Diluent 12 (consisting of Cremophor EL@ (polyethoxylated castor oil) containing 50% absolute ethanol), and paclitaxel dissolved in Diluent 12 (at a concentration of 30 m&5 ml) either were obtained from the National Cancer Institute (Bethesda, MD) or were prepared fresh from crystalline tax01 and pure solvents. Cremophor EL@ was a gift from BASF Corporation (Parsippany, NJ). Phospholipids were purchased from Avanti Polar Lipids (Birmingham, AL) or Princeton Lipids (Princeton, NJ) and stored in chloroform under argon at -70°C. All organic solvents used were reagent grade or better. Female BALB/c and DBA-2 mice were obtained from Harlan Sprague Dawley (Indianapolis, IN). 2.2. Preparation
of paclitaxel-liposomes
Paclitaxel-liposomes were prepared by hydration of a lyophilized powder consisting of paclitaxel and phospholipids, using a method described elsewhere in detail [lo]. Briefly, phosphatidylcholine and phosphatidylethanolamine were dissolved in chloroform at a 9:l molar ratio. Paclitaxel (10 mg/ml in methanol) was added to achieve a final lipid/paclitaxel molar ratio of 4O:l. The solvent was evaporated in a rotary evaporator at 4O”C, and the resulting paclitaxel/lipid film was dissolved in tert-butanol to achieve a lipid concentration of 100 mM. Two to 10 ml aliquots of the butanolic solution were placed in sterile tubes, shell-frozen in liquid nitrogen, and lyophilized for
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24 h. The lyophilized powder was hydrated in a buffer consisting of NaCl (145 mM), N-Tris[hydroxymethyl]-2-aminoethane-sulfonic acid (10 mM), and etbylenediamine tetraacetate (0.1 mM) to produce suspensions of multilamellar vesicles (MLV). Liposomes were analyzed for paclitaxel by reverse-phase HPLC [13] and for phospholipid content by phosphorus assay [14]. 2.3. Cell growth inhibition
experiments
Cells were suspended at a density of 2 x 104/ml in 24-well plates (Costar) and allowed to grow overnight before treatment. Wells in triplicate were exposed to various concentrations of paclitaxel, added either as liposomes or as free drug adsorbed to serum proteins in the absence of organic solvent. Control wells were treated with equivalent volumes of drug-free buffer or media. Cells were counted after 48 h, and the ICss (concentration resulting in 50% growth inhibition) for each concentration-effect curve was calculated graphically. 2.4. Toxicity of paclitaxel-liposomes The maximum tolerated dose (MTD) of each paclitaxel formulation was estimated by IP administration of multiple doses to healthy Balb/c female mice, using 3 animals per group. Each group was injected daily with free paclitaxel in Cremophor/ethanol or with liposomal paclitaxel. The formulations were diluted with saline and administered in a volume of 1 ml. The starting dose was 40 mg/kg, and doses were escalated in 40 m&g increments. Drug-free liposomes or the Cremophor/ethanol vehicle without paclitaxel were used as control treatments. Drug effects were determined by close observation of weight changes; the largest cumulative dose of paclitaxel causing 110% weight loss within 1 week of treatment was defined as the MTD. Animals showing weight loss > 20% were sacrificed, as weight changes of this magnitude often indicate lethal toxicity. 2.5. Animals and tumor model Female DBA-2 mice of 16-20 g were used as hosts for P388, a murine leukemia model used previously in
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paclitaxel development [ 151. The leukemia was initiated by injecting each mouse IP with lo6 viable cells in a volume of 0.5 ml. Mice were randomized into treatment groups, and treatment was started on day 1 after tumor implantation. IP injections of drug were given on 3 consecutive days. The respective vehicles (Cremophor/ethanol or phosphatidylcholine/phosphatidylglycerol liposomes without paclitaxel) were used as control treatments. In the case of Cremophor-containing formulations, the anhydrous Cremophor/ethanol vehicle (Diluent 12) initially was diluted five-fold with saline before use, similar to the clinical procedure for administering Taxole. Drug-containing formulations or vehicles were further diluted with saline and administered in a volume of 1 ml. The dose of vehicle required to administer the highest dose of Taxole tested contained the equivalent of 0.17 ml of the anhydrous Cremophor/ethanol vehicle; that quantity of vehicle was given to control animals. 2.6. End point for assessing anti-leukemia
activity
The percent increase in lifespan (%ILS) was determined as follows: %ILS = [(MDD treated-MDD control)/MDD control] x 1001 where MDD is median day of death. The control groups consisted of tumorbearing mice treated with drug-free liposomes or with Cremophor/ethanol vehicle. The survival data were analyzed for statistical significance using the Cox-Mantel test, as implemented in the program ‘Solid Tumor’ [ 161. All animal experiments were performed in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines and were approved in advance. 3. Results 3.1. Cytostatic activity of paclitaxel liposomes To investigate whether liposomal encapsulation altered paclitaxel potency, the growth inhibitory activity against P388 leukemia was compared in vitro for free paclitaxel and paclitaxel-liposomes. The two formulations were nearly equipotent, with an ICsO of 55 f 10 nM for free paclitaxel (administered without organic solvent) and an I&, of 60 + 5 nM for paclitaxel-liposomes (data not shown). Under
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similar assay conditions [lo], the ICss values for free paclitaxel on various other tumor cell lines ranged from 90 f 10 nM (for Colon-26 murine adenocarcinoma) to 1.5 f 0.7 nM (for A121a, a human ovarian carcinoma). Thus P388 leukemia showed moderate in vitro sensitivity to paclitaxel. 3.2. Toxicity of paclitaxel liposomes. The maximum tolerated dose (MTD) of paclitaxelliposomes was investigated in healthy Balb/c female mice to assist in designing a dosing regimen for antitumor experiments with P388. In previous studies, the single-dose MTD of Taxol’ (free paclitaxel administered in Cremophor/ethanol) was approx. 50 mg/kg by the IP route [8], while paclitaxel-liposomes had a MTD of 2 200 mg/kg [8,9]. The toxicity of the Cremophor vehicle itself contributed to the lower MTD of free paclitaxel. Similar MTD results were obtained here for single injections of drug. Paclitaxel-liposomes showed a cumulative MTD of 240 mg/kg when divided in 3 equal doses and given on consecutive days. In contrast, cumulative doses of only 160 mg/kg free paclitaxel in Cremophor/ethanol were lethal; with the q3d dosing scheme employed here, the MTD for free paclitaxel was approx. 120 mgJkg. Vehicle contributions to the MTD were ruled out by control experiments with vehicle alone; the quantity of drug-free liposomes or Cremophorl ethanol vehicle required to administer cumulative dose of up to 400 mgJkg paclitaxel (in equal injections given on 10 consecutive days) was not toxic upon IP administration (data not shown). 3.3. Antitumor activity The intraperitoneal P388 leukemia model has been used extensively to investigate the antitumor activity of paclitaxel [15], and was selected to evaluate the toxicity and antitumor activity of free and liposomal paclitaxel administered IP. Free paclitaxel dissolved in Cremophor/ethanol (Taxol@) was tested at cumulative doses of 40,60,X0,100 and 120 mg/kg. Paclitaxel incorporated in liposomes was tested at the same doses, and at 200 and 300 mg/kg. The total dose was administered by IP injection in 3 equal fractions on 3 consecutive days, starting on day 1 after tumor implantation. The number of mice in treatment groups
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ranged from 5 to 9. Control groups were tumor-bearing animals that were treated with saline or Cremophor/ethanol (Diluent 12) diluted 15 (v/v) with saline. The greatest increase in lifespan mediated by free paclitaxel (as the Taxol’ formulation) was observed with a dose of 80 mg/kg; mice in this group survived approximately 42% longer than did controls (Fig. la; analysis in Table 1). Mice treated with both lower (40 or 60 mg/kg; Fig. la) and higher (100 or 120 mgikg; Fig. lb) doses of free paclitaxel showed a smaller extension of lifespan compared to the group receiving 80 mg/lcg. For mice treated with 120 m&kg free paclitaxel, the lifespan was nearly equivalent to that of untreated control mice (Table 1). Animals in the 100 and 120 m&g groups appeared to have less ascites than did mice treated with 40 or 60 mg/kg (data not shown), suggesting that greater antitumor activity occurred in the high-dose groups, but was accompanied by greater toxicity. The greatest increase in lifespan mediated by paclitaxel-liposomes, approx. 40% greater than untreated controls, was observed in the 80 mg/kg group (Fig. la). At doses of 40 or 60 m&kg liposomal paclitaxel, animals showed approximately 17 or 25% ILS, respectively (Fig. la, Table 1). Paclitaxel-liposomes at doses greater than 80 mg/ kg did not enhance lifespan further; mice in the 100 and 120 mg/kg groups (Fig. lb) showed approximately 25 and 17% ILS, respectively. At the highest doses tested, 200 and 300 mg/kg, mice exhibited signs of toxicity, including reduced survival compared to the vehicle-treated control groups (Fig. lb). The lifespan was only 42% of control for mice treated at 300 mg/kg paclitaxel in liposomes. In order to test the significance of differences in lifespan among various groups, the survival data was subjected to statistical analysis by the Cox-Mantel test, as described in Section 2. Unlike median day of death (MDD) comparisons, Cox-Mantel analysis compared the data from individual animals to calculate the statistical significance of differences among treatment groups. Cox-Mantel analysis showed that the increase in lifespan mediated by both free paclitaxel and paclitaxel liposomes was significant (compared to controls) at the 40, 60, 80 and 100 mg/kg dose levels (P < 0.01). At those dose levels, the free- and lipo-
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6
8
10
12
14
16
18
20
22
Number of Days Fig. 1. Antitumor effect of free- or liposomal paclitaxel on P388 leukemia. Itttraperitoneal P388 leukemia was initiated in female DBA mice as described in Section 2. Animals were treated on 3 consecutive days with IP injections of free paclitaxel in Cremophor/ethanoI (Diluent 12) or of paclitaxel-Iiposomes (T-lip), starting on day 1 after tumor implantation. The volume injected was 1 ml. Untreated controls received equivalent volumes of saline or Diluent 12 without paclitaxel. The number of mice per group was 5-9 (n is indicated in Tables 1 and 2). For humane reasons, moribund animals were sacrificed and deaths were recorded as occurring on the following day. (A) Survival of mice treated with paclitaxel in the range of the optimal therapeutic dose; (B) survival of mice treated with paclitaxel in the range of the MTD. Inset: treatment groups; F, free drug; L, liposomal formulation. The number following the letter designation is the cumulative dose of paclitaxel, in mg/kg.
somal paclitaxel formulations showed similar dosedependent effects on lifespan, and the two formulations could not be discriminated statistically
(P > 0.05).
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Table 1 Antitumor activity of free and liposomal paclitaxel administered i.p. against i.p. P388 leukemia Treatment
Dose @wfWW
Total dose
MDD” (range)
% Incre.aaein lifespan (%ILS)
No. of mice per group
Free paclitaxel
13.3 20.0 26.1 33.3 40.0 13.3 20.0 26.7 33.3 40.0 66.7 100.0 0 0
40 60 80 00 120 40 60 80 100 120 200 300 0 0
15 (14-17) 16 (M-21) 17 (15-19) 15 (12-18) 12 (9-20) 14 (13-18) 15 (13-18) 17 (13-20) 15 (11-16) 13.5 (7-18) 9 (7-10) I (7-9) 13 (12-14) 12 (12-13)
25 33 41.7 25 0 16.7 25 40 25 16.7 -25 -41.7 8.3 0
8 9 9 7 5 7 9 9 9 8 5 5 5 5
Liposomal paclitaxel
Diluent 12 Saline
aMDD, median day of death. b%ILS, percent increase in life span.
In order to investigate the effect of doses in the range of the MTD of each formulation, free paclitaxel in Cremophor/ethanol was administered at cumulative doses of 80, 100 and 120 mgjkg, and paclitaxel-liposomes were tested at cumulative doses of 80, 100, 120, 200 and 300 m&kg. For both Cremophor-conmining- and liposomal formulations, the maximum %ILS (approx. 38% greater than controls), was observed with a cumulative dose of 80 mg/kg (Table 2). Treatment with paclitaxel-liposomes at a cumulative dose of 100 mg/kg mediated a modest
ILS (16%), while free paclitaxel at 100 mgfkg resulted in a 28% decrease in median lifespan relative to vehicle-treated controls. Treatment with paclitaxel-liposomes at 120 mgfkg resulted in a median lifespan approx. equal to that of controls, while mice treated with 120 mg./kg free paclitaxel showed a markedly reduced lifespan, 44% shorter than vehicle-treated controls. A dose of 300 m@kg liposomal paclitaxel was required to produce a reduction in lifespan that was roughly similar to the toxic effect observed with 120 mg/kg free paclitaxel (Table 2).
Table 2 Antitumor activity of high-dose paclitaxel administered i.p. against i.p. P388 leukemia Treatment Free paclitaxel Liposomal paclitaxel
Diluent 12 Blank-MLV
26.7 33.3 40.0 26.7 33.3 40.0 66.7 100.0 0.0 0.0
“MDD, median day of death. b%ILS, percent increase in life span.
Total dose
MDD” (range)
80 100 120 80 100 120 200 300 0 0
22 (21-25) 12 (8-25) 7 (7-l 1) 22 (19-23) 19 (13-23) 15 (12-24) 12 (8-24) 7 (7-8) 17 (16-19) 16 (1616)
% Increase in lifespan (%ILS)b
No. of mice per group
38 -28 -56 38 16 -06 -25 -56 cm 00
10 10 5 10 10 5 5 5 5 5
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Cox-Mantel statistical analysis showed that the increase in lifespan mediated by treatment with 80 mg/kg free or liposomal paclitaxel was significant compared to controls (P < O.OOl), but differences between the two formulations could not be discriminated statistically (P > 0.05) at that optimal dose. 4. Discussion Intraperitoneal chemotherapy has been proposed for to improve the management of cancers residing in the peritoneal cavity [ 17,181. From physiological modeling studies and from experimental data, it has been found that the clearance of some anti-neoplastic drugs from the peritoneum is considerably lower than their clearance from plasma [ 171. A blockade of lymphatic drainage may occur in diseases such as ovarian cancer [ 171, and this blockage may reduce further the peritoneal clearance of drugs with particular physical properties, or drugs associated with particulate carriers such as liposomes. For such agents, direct IP administration may result in sustained high drug concentrations in the immediate vicinity of the target site, and enhanced tumor exposure to drug. Paclitaxel appears to have pharmacokinetic properties that make it well-suited for intraperitoneal chemotherapy; after IP administration of Taxole to humans, a high paclitaxel concentration in peritoneal fluids was sustained for long periods [3]. However, severe abdominal pain was dose-limiting, and precluded dose escalation. Drug or vehicle could contribute to the toxicity, and mechanisms likely include direct irritation and chemical peritonitis, analogous to the injection-site vesicant action observed previously [S] . Liposomal encapsulation of erosive drugs such as doxorubicin has been shown to reduce local drug toxicity without a significant impact on antitumor effect [ 11,121, and previous work showed liposomes to be a means to eliminate the Cremophor and ethanol excipients that are used in the Taxol@ formulation [6-lo] of paclitaxel. For drug administered intravenously, liposomal formulations have reduced both the acute, vehicle-dependent toxicity [ 19-221 and the delayed, mechanism-dependent toxicity of paclitaxel itself. The results presented here demonstrate that paclitaxel liposomes administered IP also had lower cumulative toxicity in tumor-bearing mice than did the paclitaxel-
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Cremophor formulation used clinically, and the acute, vehicle-mediated toxicity was eliminated. Although the optimal dose for extending lifespan was similar for both the free- and liposomal paclitaxel formulations, and was achieved at doses significantly lower than the MTD of either formulation, the higher MTD of the liposomal formulation may nonetheless confer clinical advantage. The P388 tumor is moderately sensitive to paclitaxel, and responds to the conventional Cremophor-containing formulation. However, tumors may have significantly greater resistance to the drug, or may acquire resistance through treatment. Previously we compared the antitumor effect of free- and liposomal paclitaxel administered IV against Colon-26 [9], a resistant tumor cited as non-responsive to Taxole Rose, 1992 #154. Growth delay of Colon-26 was not observed with any dose of free paclitaxel (Taxole), including doses having delayed lethality [9]. Growth delay was only observed with liposomal paclitaxel, at doses that would be 100% lethal if given in the Cremophor/ethanol vehicle [9]. Liposomal formulations were well-tolerated, with no drug-mediated lethality observed. Overall, it may be necessary clinically to accept some degree of toxicity in order to exert control of tumor growth, and the liposomal formulation tested here allows the administration of higher paclitaxel doses for a given degree of toxicity. The mechanism by which liposomal encapsulation elevates MTD without altering antitumor effect is not clear. In vitro testing showed that free- and liposomal paclitaxel were equipotent, arguing against a loss of potency in the liposome-based formulation. Alternative hypotheses deserve further exploration, including the possibility that encapsulation in liposomes alters the pharmacokinetics of paclitaxel, and that the pharmacokinetic profiles that mediate toxicity to tumor versus critical tissues are different. The results also suggest that paclitaxel is released from the liposomes to exerts its effect as free drug; the rapidly-growing P388 cells occlude the lymphatic drainage of the peritoneal cavity, and likely restrict the liposomes to the peritoneum. However, the site of dose-limiting toxicity likely is reached through the systemic circulation. If comprehensive pharmacokinetic experiments confirm such an hypothesis, then further alteration of the liposomal vehicle may be possible to enhance the paclitaxel antitumor effect. It may be possible to
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develop liposomes having further-reduced rates of paclitaxel release, thereby enhancing the residence time of drug in the tumor-containing compartment. In conclusion, the experiments presented here suggest that encapsulation of paclitaxel in liposomes may allow the administration of the drug with lower toxicity than observed clinically for equivalent doses of Taxol@, or may allow the administration of higher paclitaxel doses with toxicity equal to that which is accepted currently. Given the interest in treating patients at doses greater than the maximum tolerated dose, which necessitates ‘rescue’ with granulocyte colony-stimulating factor (G-CSF) [23,24], it is possible that the reduction in toxicity achieved by encapsulation in liposomes could reduce or obviate the need for cytokine rescue. The results also underscore the possibility that with some tumors, higher doses of paclitaxel may enhance toxicity without improving overall antitumor efficacy [25]. Finally, a comparison of the liposome-based formulation with the solventbased Taxol@ formulation suggests the intriguing possibility that alterations in paclitaxel formulation may decouple toxicity and antitumor effect. An elucidation of the mechanistic basis for this decoupling could hold considerable benefits to patients, should it be possible to manipulate independently the dose-responses for toxicity and antitumor effect. Acknowledgements Paclitaxel was supplied by the National Cancer Institute, Bethesda, MD. We are indebted to Dr. Matt Suffness (recently deceased), National Cancer Institute, and Dr. Eric Mayhew, now at The Liposome Company (Princeton, NJ) for their enthusiasm, advice and encouragement. This work was supported by grant CA55251 from the National Cancer Institute, National Institutes of Health. References [l]
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Donehower, R. (1993) A phase I and pharmacologic study of paclitaxel and cisplatin with granulocyte colony-stimulating factor: neuromuscular toxicity is dose-limiting, J. Clin. Oncol., 11, 2010-2020. [24] Seidman, A., Reichman, B., Crown, J., Yao, T., Heelan, R., Hakes, T., Lebwohl, D., Gilewski, T., Surbone, A., Currie, V., Hudis, C., Klecker, R., Jam&Dow, C., Collins, J., Quinlivan, S., Berkery, R., Toomasi, F., Canetta, R. and Norton, L. (1993) Tax01 plus recombiit human granulocyte-colony stimulating factor as initial and as salvage chemotherapy for metastatic breast cancer: a preliminary report, J. Natl. Cancer Inst. Monographs, 15, 171-176. [25] Markman, M. (1993) Optimal versus maximally tolerated dose in cancer chemotherapy treatment, J. Cancer Res. Oncol., 119, 576-577.