Abstract
This study investigates 1) the anticancer efficacy of a new squalenoyl prodrug of gemcitabine (SQgem) in nanoassembly form compared with gemcitabine at equitoxic doses and 2) the subacute and acute preclinical toxicity of these compounds. The toxicity studies revealed that SQgem nanoassemblies, like gemcitabine, were toxic, and they led to dose-dependent mortality after daily i.v. injections for 1 week, irrespective of the route of administration. However, a 4- to 5-day spaced dosing schedule (injections on day 0, 4, 8, and 13) was proved to be safer in terms of weight loss and hematological and other toxicity. Using this spaced dosing schedule, SQgem nanoassemblies exhibited impressive anticancer activity in mice bearing L1210 leukemia because this treatment led to 75% long-term survivors. In contrast, at equitoxic doses, neither free gemcitabine nor cytarabine led to longterm survivors and all the mice of these groups died of the disease. Further toxicity studies performed at lethal doses by blood and serum analysis and organ weight determinations revealed that the hematological toxicity was the dose-limiting toxicity in both SQgem nanoassemblies and gemcitabine, whereas probable gastrointestinal toxicity was also associated with free gemcitabine. The SQgem nanoassemblies did not display hepatotoxicity, which is one of the clinically encountered toxicities of gemcitabine. To summarize, these preclinical studies demonstrated that the toxicological profile of new squalenoyl gemcitabine nanomedicine was not distinct from that of the parent gemcitabine, whereas it was much more potent than gemcitabine at equitoxic doses and cytarabine at clinically relevant doses. These data support the candidature of SQgem for clinical trials.
Gemcitabine is an anticancer nucleoside analog indicated in the clinic for the treatment of various solid tumors. Although the anticancer activity of gemcitabine against leukemia has been demonstrated in preclinical models in which the leukemia cells were introduced intraperitoneally (Hertel et al., 1990), it is not indicated in the clinic for this purpose. Furthermore, these preclinical models do not mimic the real clinical situations. In addition, gemcitabine was recently shown to be insufficiently active against leukemia in phase II clinical trials (Angiolillo et al., 2006, Wagner-Bohn et al., 2006). We have recently developed a new prodrug of gemcitabine by conjugation with squalene (squalenoylation) (Couvreur et al., 2006a). The linkage of squalene was performed at the level of the 4-amino group of gemcitabine, rendering the molecule more amphiphilic, and resulting in their spontaneous aggregation as nanoassemblies of approximately 130 nm in diameter. These squalenoyl gemcitabine (SQgem) nanoassemblies were demonstrated to prevent the deamination of gemcitabine in vitro, thus overcoming one of the main drawbacks to its use, its short biological half-life (Couvreur et al., 2006b). In addition, in vitro, the SQgem nanoassemblies were demonstrated to be more cytotoxic than gemcitabine toward various resistant leukemia cell types and even to reverse resistance. In preliminary investigations, it was found that these nanoassemblies displayed better anticancer activity as well as high level of induction of apoptosis than gemcitabine, against systemically developed leukemia at lower equivalent doses of gemcitabine (Reddy et al., 2007). However, it rapidly became obvious that it would be more meaningful to compare the anticancer activity of the new SQgem nanomedicine with gemcitabine at equitoxic doses, to clearly demonstrate which was the more efficient.
After i.v. administration, the dose-limiting toxicity of gemcitabine has been reported to be its hematological toxicity, which varies with the administration schedule. This includes neutropenia, anemia, and thrombocytopenia. The dose-limiting toxicity was myelosuppression, with thrombocytopenia and anemia quantitatively more important than granulocytopenia (Abbruzzese et al., 1991). Apart from this, hepatotoxicity is also encountered clinically after treatment with gemcitabine (Robinson et al., 2003). At the preclinical level, after proof of efficacy, the determination of toxicity and safety levels of new anticancer compounds are equally important prerequisites for submission to clinical trials.
Thus, in the present work, we have determined the preclinical efficacy of SQgem in comparison with the gemcitabine at equitoxic doses, and we performed comparative toxicological assays of both drugs at low doses, equitoxic doses, and also at toxic doses. The anticancer activity of the SQgem nanoassemblies was also compared with the clinically indicated antileukemia agent cytarabine administered according to various schedules.
Materials and Methods
Gemcitabine hydrochloride was purchased from Sequoia Research Products Ltd. (Oxford, UK). Squalene and dextrose were purchased from Sigma-Aldrich Chemical Co. (Paris, France).
Synthesis and Characterization of SQgem. SQgem was synthesized by covalent coupling of 1,1′,2-trisnorsqualenic acid onto the 4-amino group of the cytosine moiety of gemcitabine, and it was characterized as reported previously (Couvreur et al., 2006a). Practically, to a stirred solution of trisnorsqualenoic acid (0.50 g; 1.2 mmol) in anhydrous tetrahydrofuran (3 ml) we added, under nitrogen, triethylamine (0.15 g; 1.4 mmol). The mixture was cooled to –15°C, and a solution of ethylchloroformate (0.135 g; 1.2 mmol) in anhydrous tetrahydrofuran (3 ml) was added dropwise. The mixture was stirred at –15°C for 15 min, and a solution of gemcitabine hydrochloride (0.37 g; 1.2 mmol) and triethylamine (0.15 g; 1.4 mmol) in anhydrous dimethylformamide (5 ml) was added dropwise to the reaction mixture at the same temperature. The reaction mixture was stirred for 72 h at room temperature, and then it was concentrated in vacuo. Aqueous sodium hydrogen carbonate was added, and the mixture was extracted with ethyl acetate (3 × 50 ml). The combined extracts were washed with water, dried over MgSO4, and concentrated under reduced pressure. The crude product was purified by chromatography on silica gel by eluting with 1 to 5% methanol in dichloromethane containing 0.5% triethylamine to obtain 57% yield of pure 4-(N)-trisnorsqualenoylgemcitabine as a white amorphous solid.
Preparation and Characterization of SQgem Nanoassemblies and Squalene Nanoparticles. SQgem nanoassemblies were prepared by nanoprecipitation technique. In brief, an ethanolic solution of SQgem (10 mg/ml) was added dropwise under stirring (500 rpm) to a 5% aqueous dextrose solution. Precipitation of the SQgem nanoassemblies occurred spontaneously. Ethanol was completely evaporated using a Rotavapor (BUCHI Sarl, Rungis, France) to obtain an aqueous suspension of pure SQgem nanoassemblies. Nanoparticles made of squalene alone were prepared in a similar manner. An ethanolic solution of squalene (10 mg/ml) was added dropwise under stirring in a 5% aqueous dextrose solution containing Pluronic F68 [1% (w/v)]. Ethanol was completely evaporated using a Rotavapor to obtain the aqueous suspension of pure squalene nanoparticles.
The mean diameter of the squalene or SQgem nanoassemblies was determined at 20°C by quasi-elastic light scattering with a nanosizer (Coulter N4MD; Beckman Coulter, Inc., Fullerton, CA).
Subacute and Acute Toxicity Studies. The toxicity studies were performed following U.S. Food and Drug Administration guidelines (http://www.cfsan.fda.gov/~redbook/red-ivb1.html). The animal experiments were carried out according to the principles of laboratory animal care and legislation in force in France. Toxicity studies were performed on healthy DBA/2 mice (5–6 weeks old) following a schedule of daily injections of SQgem nanoassemblies or gemcitabine by i.v., i.m., or s.c. routes. Untreated mice were used as a control. The mice treated with squalene nanoparticles (100 mg/kg) were used as a placebo-treated control. The mice were regularly monitored for differences in weights and survival.
A four-dose schedule with 4 to 5-day spacing (injections on day 0, 4, 8, and 13) was also adopted for SQgem nanoassemblies and gemcitabine by the i.v. route. After injection of the formulations, the mice were regularly monitored for differences in weights and survival. Subsequently, the safety of SQgem nanoassemblies was evaluated in comparison with gemcitabine using a four-dose spacing schedule (i.v. injection on day 0, 4, 8, and 13) at maximal tolerated doses (MTD); blood and serum parameters (from pooled serum samples) (n = 8 mice) were monitored in comparison with the untreated group.
Finally, an assay was performed at toxic doses for both SQgem nanoassemblies (30 mg/kg equivalent of gemcitabine) and gemcitabine (5 × 15 mg/kg), and the prevalent toxicity was determined by measuring the blood and serum parameters and organ weights.
In Vivo Anticancer Activity of SQgem Nanoassemblies Compared with Gemcitabine. DBA/2 mice (5–6 weeks old), weighing approximately 15 to 18 g, were used for the study. The mice were provided with standard mouse food and water ad libitum. The L1210 wt leukemia cells were maintained in vitro, and they were injected intravenously (1 × 105) into the mice, to develop a systemic metastatic leukemia model.
The mice were divided into six groups of seven to eight mice each: untreated, treated with squalene nanoparticles, treated with 100 mg/kg gemcitabine, treated with 20 mg/kg equivalent SQgem nanoassemblies in gemcitabine, treated with 100 mg/kg cytarabine, and treated with 100 mg/kg cytarabine every day for 5 days. After injection of leukemia cells (day 0), all groups of mice received the treatment by i.v. injection on days 1, 5, 9, and 14 (i.e., days after injecting leukemia cells), with the exception of the untreated group and the group treated with cytarabine daily by the i.v. route. The mice were monitored regularly for weight differences and survival.
Statistical Analysis. Statistical analysis was performed using Student's t test and one-way analysis of variance wherever relevant using GraphPad software (GraphPad Software Inc., San Diego, CA). The survival data were analyzed using the Kaplan-Meier test to determine the level of significance. Median survival time (MST) was calculated from the survival data using MedCalc version 9.3.9.0 software (MedCalc Software, Mariakerke, Belgium). Increase in life span (ILS) was calculated using the following formula (Ojo-Amaize et al., 2007): ILS (%) = [(MST of treated group – MST of untreated group)/MST of untreated group] × 100.
Results
The first toxicity assays were carried out using daily dosage for 1 week, starting from the lower doses. The doses were from 2.5 to 10 mg/kg Eq gemcitabine injected intravenously into healthy mice. After injections, the mice were monitored for weight loss and survival. The overall body weight loss was in the following order: 10 mg/kg SQgem nanoassemblies > 5 mg/kg SQgem nanoassemblies > 10 mg/kg Gem > 2.5 mg/kg SQgem nanoassemblies > 5 mg/kg Gem > 2.5 mg/kg Gem, and values ranged between 15 and 22% for both SQgem nanoassemblies and gemcitabine (Fig. 1A). When evaluated on day 4, the body weights of all treated groups except those given 2.5 mg/kg Gem and 5 mg/kg Gem were significantly lower (p < 0.05) than those of the untreated group. After the completion of injections, 40% mice of SQgem nanoassemblies-treated group and all mice of 2.5 mg/kg Gem-treated group recovered from the body weight loss. From the survival curve, it is evident that except in the groups treated with 2.5 mg/kg Gem or 2.5 mg/kg Eq SQgem nanoassemblies, all mice in other groups of gemcitabine and SQgem nanoassemblies treatments did not survive the treatment (Fig. 1B).
To investigate whether the toxicity depended on the route of administration, the 10-mg/kg dose of either gemcitabine or the equivalent in SQgem nanoassemblies (which caused higher toxicity) was administered by the i.m. and s.c. routes. When evaluated on day 3, the body weight losses of all the groups treated with SQgem nanoassemblies were considerably lower than those of the Gem-treated groups (p < 0.05) (Fig. 2A). Regardless of the injection route, both Gem and SQgem nanoassemblies were fatal to all mice at this dose (Fig. 2B). Because s.c. and i.m. administration did not reveal any advantage in terms of toxicity over the i.v. route, which is the route of injection for gemcitabine in the clinic, this mode of injection was retained for further toxicity studies. These studies were performed following a spacing schedule by injections of either gemcitabine or SQgem nanoassemblies on day 0, 4, 8, and 13. In preliminary investigations, the lower doses of SQgem nanoassemblies (up to 15 mg/kg Eq gemcitabine) or gemcitabine (up to 40 mg/kg) did not show toxicity in terms of body weight loss or survival; hence, only higher doses were evaluated further. After i.v. injection of gemcitabine at doses of 50, 100, and 120, and 300 mg/kg, the highest dose caused considerable body weight loss (p < 0.05 at all the time points evaluated, starting from day 3 of first injection) (Fig. 3A) compared with that of the untreated group and complete mortality, whereas 120 mg/kg was determined as the lethal dose 10% (LD10) and 100 mg/kg was considered as the maximal tolerated dose, which did not cause any mortality and a minimal body weight loss (Fig. 3B). In the SQgem nanoassemblies, of the four doses tested, i.e., 15, 20, 22, and 30 mg/kg Eq, the highest dose provoked a considerable decrease in body weight (ranging between 5 and 25%) (p < 0.05, at all the time points evaluated starting from day 6 of first injection) (Fig. 4A) compared with that of the untreated group and complete mortality (Fig. 4B), whereas 22 mg/kg Eq was found to be the LD10 (body weight loss up to 10%). In contrast, a dose of 20 mg/kg did not cause any mortality, and the difference in the body weights was also minimal; hence, this dose was considered as the maximal tolerated dose.
Therefore, we used the 0-, 4-, 8-, and 13-day i.v. injection schedule (in the mice receiving injections with leukemia cells on day 0, the days of treatment became 1, 5, 9, and 14), at the MTD in L1210 wt leukemia-bearing mice to compare the anticancer efficacy of the SQgem nanoassemblies with that of gemcitabine. Cytarabine was also tested as a reference for the treatment of leukemia in clinical practice (Ravindranath, 2003; Ribera and Oroil, 2007; Roboz, 2007), at a dose similar to that of gemcitabine (4 × 100 mg/kg on day 1, 5, 9, and 14). In addition, because the clinical schedule of administration is the daily dosing schedule for five consecutive days, this schedule was also used (5 × 100 mg/kg each day for 5 days). The toxicity was evaluated by whole blood and serum analysis following the same dosage and administration schedule. The untreated L1210 wt leukemia-bearing mice died after 14 to 18 days, whereas the mice treated with the placebo squalene nanoparticles (at a dose as high as 100 mg/kg) showed no differences in survival compared with the untreated mice (Fig. 5). Cytarabine injected every day or according to the day 1, 5, 9, and 14 schedule, and gemcitabine, significantly improved the survival of the leukemia-bearing mice compared with that of the untreated group (p < 0.05) (Fig. 5). Noteworthy, the gemcitabine treatment led to a greater increase in life span (% ILS = 150) compared with the cytarabine treatments (% ILS = 43.3 and 40, respectively) in the leukemia-bearing mice (Table 1). In addition, the T/U ratio (treated/untreated ratios calculated from the median survival times) of the gemcitabine-treated group was higher (∼1.7-fold) than those of cytarabine-treated groups (Table 1). The SQgem nanoassemblies were very much more efficient at MTD compared with that of untreated group (p = 0.0001), placebo squalene-treated group (p < 0.0001), cytarabine-treated groups (p < 0.005), and Gem-treated group (p < 0.005) as analyzed using Kaplan-Meier test, and they allowed 75% long-term survivors (Fig. 5).
Blood and serum analyses were performed 5 days after the end of the day 0, 4, 8, and 13 i.v. injection schedule of either SQgem nanoassemblies or gemcitabine at their MTD in healthy mice. No considerable differences in either blood parameters (Table 2) or serum parameters (Table 2) were observed for SQgem nanoassemblies and gemcitabine compared with the untreated group. It is noteworthy that the levels of liver enzymes, such as alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase, of SQgem nanoassemblies-treated group showed no important difference from those of the gemcitabine-treated and untreated groups.
Because both SQgem nanoassemblies and gemcitabine displayed considerable toxicity and mortality at both a low-dose daily i.v. injection schedule and a high-dose day 0, 4, 8, and 13 i.v. injection schedule (doses above MTD), it was interesting to investigate the reasons behind this toxicity. Therefore, healthy mice received i.v. injections with lethal doses of SQgem nanoassemblies or gemcitabine, and the whole blood and serum parameters were evaluated as described previously. In both the treated groups, the red blood cell (RBC) count, hemoglobin, and hematocrit (HCT) did not differ much from the untreated control group; however, lymphopenia and granulocytopenia were evident in the gemcitabine-treated group (p < 0.05), whereas only lymphopenia occurred in the SQgem nanoassemblies-treated group (p < 0.05) (Table 3). In contrast, the platelet count was considerably lower in the SQgem nanoassemblies-treated group (p < 0.05) (suggesting the probability of thrombocytopenia), compared with the gemcitabine-treated and untreated group. The granulocyte depletion was noticeably less with SQgem nanoassemblies treatment (p < 0.05) compared with the gemcitabine treatment. The serum creatinine levels were found to be elevated with both SQgem nanoassemblies treatment and gemcitabine treatment; however, a noticeable elevation of serum amylase and lipase levels was observed in the gemcitabine-treated group (p < 0.05) but not in the SQgem nanoassemblies-treated group (Table 3) compared with the untreated control group.
The weights of organs were also evaluated following the same lethal dose injections of SQgem nanoassemblies or gemcitabine. A significant decrease in spleen and kidney weights occurred in the gemcitabine-treated group (p < 0.05), whereas only spleen weight was found lower in SQgem nanoassemblies-treated group compared with the untreated mice (p < 0.05) (Fig. 6). The weights of liver and heart were not greatly affected in either group compared with the untreated group.
Discussion
The subacute and acute toxicological evaluation of SQgem nanoassemblies was started using low doses. Following a daily dosing schedule for 1 week, both SQgem nanoassemblies and gemcitabine displayed dose-dependent toxicity in terms of both body weight loss and survival. This toxicity was similar regardless of the mode of injection, suggesting that both SQgem nanoassemblies and gemcitabine were well absorbed after i.m. or s.c. administration. However, spacing the injections reduced toxicity and mortality of the animals, thus providing better tolerance compared with the daily dosing schedule. Improvement in the cytotoxicity of gemcitabine has previously been reported following prolonged i.v. injection rather than bolus injection (Braakhuis et al., 1995; Tempero et al., 2003). This may be due to an improved accumulation of the active triphosphate form of gemcitabine (difluoro deoxycytidine triphosphate) inside the cells (Grunewald et al., 1990; Abbruzzese et al., 1991). The same phenomenon could explain the increased toxicity of both SQgem nanoassemblies and gemcitabine following daily injection schedule, because frequently repeated administration of these drugs would lead to higher and sustained plasma levels, thereby overloading the targeted organs and cells by accumulation of cytotoxic difluorodeoxycytidine triphosphate. In contrast, spaced dosing would prevent this phenomenon by avoiding toxic accumulation of these drugs in the targeted organs, thereby reducing the toxicity. Thus, in SQgem nanoassemblies and gemcitabine, the spacing schedule is safer than the daily dosing schedule. The subsequent question to be answered is whether the spacing schedule, although safer, can provide adequate anticancer activity in vivo at the MTD. Thus, this dose schedule was used to evaluate the anticancer activity of gemcitabine and SQgem nanoassemblies on L1210 leukemic mice (in mice receiving injections with leukemia cells on day 0, the days of treatment became 1, 5, 9, and 14). Because cytarabine is a clinically indicated antileukemic drug (Ravindranath, 2003; Ribera and Oroil, 2007; Roboz, 2007), this compound was also used in this study as a reference. It was observed that cytarabine, although improved the survival of the mice, showed considerably lower anticancer activity than gemcitabine and SQgem nanoassemblies. The percentage of ILS of treated groups was in the following order: cytarabine (day 1, 5, 9, and 14 day injection schedule) < cytarabine (injections on five consecutive days) < Gem (MTD). The median survival time and ILS could not be calculated for SQgem nanoassemblies-treated group because it resulted in a very high percentage of long-term survivors (75%). The significantly greater anticancer activity at MTD of SQgem nanoassemblies compared with both gemcitabine and clinically indicated cytarabine against L1210 wt leukemia, which is generally considered as a relatively resistant preclinical model, demonstrates the potential of SQgem nanoassemblies for leukemia treatment. The absence of activity and toxicity of squalene nanoparticles even at higher doses clearly shows that the observed anticancer activity of the SQgem nanoassemblies was solely due to its conjugate nature and that it did not include a contribution from squalene itself. This impressive anticancer activity of gemcitabine when conjugated with squalene in nanoassembly form is probably due to protection against deamination and prolonged release of the active compound inside the cells. However, the mechanism behind this remarkable observation requires further investigation at the cellular level. The absence of hematological toxicity of SQgem nanoassemblies as evaluated by blood and serum parameters indicates the safety of this administration schedule with regard to the main target toxicity of gemcitabine. It should also be noted that the SQgem nanoassemblies did not cause any hepatotoxicity, another clinically encountered toxicity after treatment with gemcitabine.
Finally, the mice received injections with lethal doses of both gemcitabine and SQgem nanoassemblies to investigate the reasons of the observed toxicity. The hematological toxicity was clearly evident for both treatments as shown by blood counts and reduced spleen weights. Apart from this, the hyperamylasemia and hyperlipasemia in the gemcitabine-treated group suggest probable gastrointestinal toxicity of gemcitabine, which was not observed with SQgem nanoassemblies. Together, these data suggest that the new squalenoyl gemcitabine nanomedicine has no distinct toxicological profile apart from the differences in serum gastrointestinal enzyme levels and that it is safer and impressively active when administered using a spacing schedule. This clearly indicates that this nanomedicine retains a similar toxicological profile to gemcitabine, while exhibiting an impressively greater anticancer activity against experimental leukemia.
At equitoxic doses, the SQgem nanomedicine was impressively effective in enhancing the survival time of leukemia-bearing mice, and it also led to long-term survivors, unlike the clinically indicated cytarabine and gemcitabine. This argues strongly for the candidature of this squalenoyl nanomedicine for clinical trials.
Acknowledgments
We thank Dr. G. Barratt for suggestions in preparing the manuscript.
Footnotes
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This study was supported by Agence Nationale de la Recherche Grant SYLIANU and Centre National de la Recherche Scientifique Grant Ingénieur de valorization. L.H.R. was supported by a postdoctoral fellowship from Université Paris-Sud.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.133751.
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ABBREVIATIONS: SQgem, squalenoyl gemcitabine; MTD, maximal tolerated dose(s); wt, wild type; MST, median survival time; ILS, increase in life span; Gem, gemcitabine; T/U, treated/untreated; RBC, red blood cells(s); HCT, hematocrit.
- Received October 30, 2007.
- Accepted February 6, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
References
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