Preclinical efficacy studies of a novel nanoparticle-based formulation of paclitaxel that out-performs Abraxane
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Poly-(γ-l-glutamylglutamine)–paclitaxel (PGG–PTX) is a novel polymer-based formulation of paclitaxel (PTX) in which the PTX is linked to the polymer via ester bonds. PGG–PTX is of interest because it spontaneously forms very small nanoparticles in plasma. In mouse models, PGG–PTX increased tumor exposure to PTX by 7.7-fold relative to that produced by PTX formulated in Cremophor. In this study, the efficacy of PGG–PTX was compared to that of Abraxane, an established nanoparticular formulation of PTX, in three different tumor models.
Efficacy was quantified by delay in tumor growth of NCI H460 human lung cancer, 2008 human ovarian cancer and B16 melanoma xenografts growing in athymic mice following administration of equitoxic doses of PGG–PTX and Abraxane administered on either a single dose or every 7 day schedule. Toxicity was assessed by change in total body weight.
The efficacy and toxicity of PGG–PTX was shown to increase with dose in the H460 model. PGG–PTX was ~1.5-fold less potent than Abraxane. PGG–PTX produced statistically significantly greater inhibition of tumor growth than Abraxane in all three tumor models when mice were given single equitoxic doses of drug. When given every 7 days for 3 doses, PGG–PTX produced greater inhibition of tumor growth while generating much less weight loss in mice bearing H460 tumors.
PGG–PTX has activity that is superior to that of Abraxane in multiple tumor models. PGG–PTX has the potential to out-perform Abraxane in enhancing the delivery of PTX tumors while at the same time further reducing the toxicity of both single dose and weekly treatment regimens.
KeywordsPaclitaxel Abraxane Drug delivery Lung cancer Ovarian cancer Melanoma
Maximum tolerated dose
70 kDa PGA to which both additional glutamine side chains and PTX have been added
Paclitaxel (PTX) is effective for the treatment of a wide variety of cancers but, because of its limited solubility in water, it is currently formulated as a concentrated solution containing 6 mg PTX/ml Cremophor EL and ethanol (50% v/v) that must be further diluted before administration. Cremaphor EL is a biologically and pharmacologically active compound and its use is associated with acute hypersensitivity reactions [4, 15]. Many investigators have tried to develop PTX formulations using liposomes, microspheres, micelles, nanoparticle, prodrugs, and polymer-drug conjugates [4, 24]. One of these, paclitaxel protein-bound particles for injection (Abraxane), is based on the use of a nanoparticle made from a mixture of paclitaxel and albumin and is now marketed for the treatment of breast cancer. However, the fractional improvement in breast cancer progression-free survival was quite modest . Another, CT-2103 (Xyotax), takes advantage of the fact that paclitaxel can be made more soluble by conjugating it to the water-soluble polymer poly(l-glutamic acid) [9, 10, 22]. Like Abraxane, CT-2103 exhibits reduced toxicity and increased efficacy in preclinical models including paclitaxel-resistant tumors [1, 2, 3, 6, 7, 11, 12, 13, 16, 18, 25, 26]. However, despite favorable phase II clinical trial results [13, 21, 23], three randomized phase III trials in patients with non-small cell lung cancer failed to demonstrate an improvement in either progression-free or overall survival [8, 17, 19] and CT-2103 has not yet received marketing approval.
The efficacy of PGG–PTX has now been tested in a panel of murine and human tumor xenografts in which the activity of PGG–PTX was directly compared to that of Abraxane when the two drugs were given at equitoxic doses. We report here that PGG–PTX out-performs Abraxane in several of these models on a single dose schedule, and that PGG–PTX is substantially less toxic than Abraxane when used on a multidose schedule.
Materials and methods
Chemicals and reagents
Poly-(γ-l-glutamylglutamine)–paclitaxel (PGG–PTX) was synthesized from commercially available poly(l-glutamic acid) sodium salt with a molecular weight of 24,880 Da. l-glutamic acid di-t.-butyl ester hydrochloride was linked to each monomer in the polymer using a coupling reagent. PTX was then conjugated to the poly(l-glutamylglutamate) using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide. The PTX content of PGG–PTX was quantified and found to be 35.8% (w/w). The PGG–PTX was dissolved in saline at concentration of 17.5 mg PTX/ml (50 mg total weight/ml). Fresh stock solutions were prepared on the day of injection. Commercially available Abraxane (Los Angeles, CA) was dissolved in saline at concentration of 80 mg/ml. All other chemicals and reagents were obtained from Sigma Chemical Co. (St. Louis, MO), Invitrogen/Life Technologies, Inc. (Carlsbad, CA), Millipore Coorporation (Temecula, CA), or Hyclone (Logan, UT).
NCI-H460 and 2008 cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum containing 100 U/ml penicillin and 100 μg/ml streptomycin. B16-F0 cells were grown in DMEM supplemented with 10% bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were split 48 h before inoculation into mice so that they were in log phase growth when harvested.
NCI-H460 and 2008 cells were suspended in serum-free RPMI 1640 medium, and B16-F0 cells suspended in serum-free DMEM medium, and then injected subcutaneously. The inoculum size per site was 4 × 106 cells for the NCI-H460 cells, 2 × 106 for the 2008 cells, and 0.5 × 106 for the B16 cells. NCI-H460 and B16 cells were injected over each shoulder and each hip of 6- to 8-week-old female athymic nude (nu/nu) mice (Charles River lab, Willington, MA) (4 sites per mouse). The 2008 cells were injected only over both shoulders. Tumors were allowed to grow until they reached an average volume of 30–40 mm3. Tumor size in mm3 was estimated from the formula (w2 × l)/2 where “l” is the longest diameter of the tumor and “w” is the diameter perpendicular to the longest diameter measured in millimeters. Tumor-bearing animals were then randomly sorted into 3 groups of 6–10 mice each. Both Abraxane and PGG–PTX were administered as a single IP injection. Control animals were injected with saline in a volume equivalent to the volume of PGG–PTX. The MTD was defined as the dose that produced 10% weight loss. Mice were weighed daily and tumor volumes were measured every other day until total tumor burden reached 1.5 cm3 at which time mice were killed. All in vivo studies were performed at the animal facility of University of California San Diego in accordance with institutional guidelines set out by Institutional Animal Care and Use Committee (IACUC).
The slope of the regression of log (tumor volume) on time was determined for each individual tumor and the mean of the slopes of all tumors within a group was compared using the Student’s t test.
Activity of PGG–PTX in NCI-H460 human lung cancer xenograft model as a function of dose
Relative efficacy of PGG–PTX and Abraxane in the NCI-H460 human non-small cell lung cancer xenograft model
Figure 3b shows that Abraxane at a dose of 250 mg/kg produced a 15 ± 6% (SEM) loss of weight with a nadir on day 5. PGG–PTX at a dose of 300 mg PTX/kg produced the same degree of weight loss (13 ± 6%) with a nadir day 6. No animals died of toxicity in the PGG–PTX group whereas there was 1 toxic death among the 6 animals in the Abraxane group. However, while both drugs produced equivalent acute reductions in body weight, weight recovery was more rapid following administration of Abraxane. Mice treated with Abraxane regained their initial body weight by day 8–9, whereas mice treated with PPG-PTX required >20 days. This difference is consistent with the observation that PGG–PTX has a much longer plasma half-life (293 h) in mice than Abraxane (19 h) in the rat [26, 27], and suggests that the PTX delivered by PGG–PTX remains in both the tumor and normal tissues for very prolonged periods of time relative to that delivered by Abraxane.
Relative efficacy of PGG–PTX and Abraxane in the human ovarian 2008 xenograft model
Relative efficacy of PGG–PTX and Abraxane in the murine B16 melanoma model
Relative efficacy of PGG–PTX and Abraxane in the NCI-H460 model using a multidose schedule
Poly-(γ-l-glutamylglutamine)–paclitaxel (PGG–PTX) is of interest as a novel drug delivery system for PTX for several reasons. First, it is unique in being a polymer-based system that spontaneously forms nanoparticles in aqueous environments. Ongoing molecular modeling studies suggest that the hydrophobic interactions between the PTX molecules randomly distributed along the PGG polymer cause collapse of the polymer strand to form a particle with a hydrophilic surface and a hydrophobic core. Second, the size of the nanoparticles as determined by dynamic light scattering is substantially smaller than those in the Abraxane formulation (~20 vs. 80–120 nm). The large difference in size is likely to translate into important differences in the behavior of the drugs both in the plasma and in tissues. Third, pharmacokinetic studies in nu/nu mice bearing NCI-H460 tumors indicate that conjugation of PTX to the PGG polymer increased plasma and tumor Cmax, prolonged plasma half-life and the period of accumulation in tumor, and reduced washout from the tumor. The plasma exposure to total taxane produced by PGG–PTX, measured over the first 340 h after injection, was 23-fold greater than that produced by unconjugated PTX, and the tumor exposure was increased by a factor of 7.7-fold . In contrast, Abraxane increased tumor exposure by only 33% relative to that attained with Cremophor-based paclitaxel (3632 vs. 2739 nCi h/g) in the MX-1 xenograft model reported by Desai et al. . The results of the current study provide an additional reason for interest in PGG–PTX. In all 3 of the models tested in this study in which a single dose of drug was used, PGG–PTX out-performed Abraxane with respect to inhibition of tumor growth when both drugs were given at doses that produced similar degrees of acute weight loss. In addition, the magnitude of the increase in effectiveness was quite substantial and statistically significant in all 3 models. Perhaps even more importantly, when given every 7 days for 3 doses, PGG–PTX produced greater inhibition of tumor growth while at the same time causing much less toxicity than Abraxane. Even while being less effective, the 40 mg PTX/kg dose of Abraxane produced progressive weight loss and was not tolerated on a weekly schedule.
Part of the explanation for the difference in efficacy of PGG–PTX and Abraxane, and for the delayed recovery of body weight in the mice treated with a single dose of PGG–PTX, likely lies in their quite different pharmacokinetics both in the plasma and in normal and malignant tissues. PGG–PTX was found to be somewhat less potent than Abraxane with respect to acute toxicity when given on a single dose schedule. Whereas the MTD for PGG–PTX was 300–350 mg PTX/kg in all 3 models, the single dose MTD for Abraxane varied from 150 mg PTX/kg in the B16 model to 250 mg PTX/kg in the NCI H460 lung cancer model. It appears that the type of tumor modulates the toxicity of Abraxane to a greater extent than that of PGG–PTX, an effect most likely related to the specific vascular anatomy of each tumor type. The plasma half-life of Abraxane in mice has not been reported but in the rat it was found to be 19.0 h; the plasma clearance was 517 ml/h/kg . In contrast, in pharmacology studies carried out in nu/nu mice bearing NCI H460 tumors executed in parallel with the efficacy studies reported here, the plasma half-life of PGG–PTX was found to be 296.2 h and the clearance only 11.5 ml/h/kg . Whereas Abraxane increased the total tumor exposure to PTX by only 33% above that produced by an equitoxic dose of PTX formulated as Taxol in the MX1 tumor model , PGG–PTX increased exposure for the NCI H460 tumors by a factor of 7.7-fold in the NCI H460 model . Studies directly comparing the tumor exposure to PTX following injection of equitoxic doses of PGG–PTX and Abraxane in the same tumor model have yet to be performed. Nevertheless, these data are consistent with the concept that PGG–PTX is more efficient at targeting PTX to tumors than Abraxane largely due to differences in their pharmacokinetics.
Abraxane has an established role in the treatment of breast cancer. We conclude from these studies that PGG–PTX has the potential to out-perform Abraxane in enhancing the delivery of PTX to such tumors while at the same time further reducing the toxicity of both single dose and weekly treatment regimens.
We thank Brian Reuter and Ho Lim Fung for technical assistance. Financial support for this work was provided by UC Discovery grant bio06-10568 and the Nitto Denko Technical Corporation. This work was supported by a public–private grant program operated by the University of California in which the University and the Nitto Denko Technical Corp each provide half of the funding. Drs. Xinghe Wang, Gang Zhao, Sang Van, Nan Jiang and Lei Yu are employees of Nitto Denko. Drs. David Vera and Stephen B. Howell are employees of the University of California, San Diego who receive research support under this grant. Drs. Vera and Howell have also served as consultants to Nitto Denko in the past.
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