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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure Statement
  8. References

The intraperitoneal administration of paclitaxel has been shown to be a promising treatment strategy for peritoneal malignancy. The present study evaluated the effects of intraperitoneal administration of NK105, a paclitaxel-incorporating micellar nanoparticle, which has been shown to have a remarkable effect in a mouse model of gastric cancer. Intraperitoneal NK105 significantly reduced peritoneal tumors in vivo compared with the conventional paclitaxel formulation of paclitaxel solubilized in Cremophor EL and ethanol (PTX-Cre). Moreover, intraperitoneal NK105 significantly reduced the size of subcutaneously inoculated tumors, whereas no such effect was seen with PTX-Cre. Similar systemic toxic effects were observed following the intraperitoneal administration of both NK105 and PTX-Cre. Although NK105 disappeared rapidly almost within a day from the peritoneal cavity, the paclitaxel concentration in peritoneal nodules 4 h after intraperitoneal administration was significantly higher in the NK105 group than in the PTX-Cre group (< 0.05), whereas there were no significant differences in liver paclitaxel concentrations between the two groups. We also evaluated the pharmacokinetics following intraperitoneal administration of NK105 and PTX-Cre. Serum paclitaxel concentrations 6, 12, 24, and 48 h after the intraperitoneal administration of the drugs were significantly higher in the NK105 than the PTX-Cre group. Furthermore, the peak serum concentration was higher in the NK105 than PTX-Cre group (24 100 ± 3560 vs 108 ± 25 ng/mL, respectively; < 0.001), as was the area under the concentration–time curve from 0 to 48 h (191 000 ± 32 100 vs 1500 ± 108 ng·h/mL, respectively; < 0.001). Therefore, intraperitoneal chemotherapy with nanoparticulate paclitaxel NK105 may offer a novel treatment strategy for improving drug delivery in gastric cancer with peritoneal dissemination because of enhanced drug penetration into peritoneal nodules and its prolonged presence in the systemic circulation. (Cancer Sci 2012; 103: 1304–1310)

Gastric cancer is the fourth leading cause of cancer-related deaths worldwide, and peritoneal dissemination is the most frequent and life-threatening form of metastasis and recurrence in patients with gastric cancer.[1, 2] The current systemic chemotherapy regimens for gastric cancer have not achieved satisfactory results, particularly in the treatment of peritoneal dissemination.[3] One of the problems with this type of therapy is the limited delivery of systemically administered drugs to the peritoneal cavity.[4] Intraperitoneal chemotherapy has been shown to be safe and effective in ovarian cancer[5-7] and is expected to provide a breakthrough in the treatment of gastric cancer with peritoneal dissemination.[8-10] However, in addition to the intraperitoneal administration of drugs, it is necessary to develop new strategies for the treatment of gastric cancer to achieve better results. Research into drug delivery systems (DDS) is ongoing and is mostly aimed at improving the penetration of drugs into tumors or prolonging the retention of drugs in the peritoneal cavity.[11, 12]

Paclitaxel, one of the most commonly used and effective anticancer agents, is not soluble in water and, for clinical use, it is conventionally solubilized using the polyoxyethylated castor oil Cremophor EL and ethanol (hereafter referred to as PTX-Cre), such as in TAXOL (Bristol-Myers Squibb, New York, NY, USA).[13, 14] The conventional PTX-Cre formulation is considered suitable for intraperitoneal chemotherapy because its larger molecular size (10–12 nm in diameter) compared with other water-soluble anticancer drugs results in prolonged retention of the drug in the peritoneal cavity.[15] However, the Cremophor EL in the PTX-Cre formulation causes severe hypersensitive reactions, including anaphylaxis, in 2–4% of patients, even after premedication with antiallergic agents.[13, 14]

Since the discovery of selective accumulation by passive targeting, also known as the enhanced permeability and retention (EPR) effect,[16] various nanoparticulate drugs have been developed for cancer treatment.[17-19] The EPR effect is based on the special characteristics of solid tumor tissues (i.e. incomplete vascular architecture, hyperpermeability of vessel walls, and incomplete lymphatic drainage).[20] Through the EPR effect, anticancer drugs incorporated into polymeric nanomicelle carriers (20–100 nm in diameter) are retained for a long period in the systemic circulation,[21, 22] are easily extravasated from tumor vessels into the interstitium of tumor tissue, and accumulate there for longer periods than conventional small molecule agents.[16, 23] Various types of Cremophor EL-free formulations, including nanoparticle paclitaxel, have recently been investigated to reduce the risk of allergic reactions and to take advantage of the EPR effect.[24-28] For example, Abraxane (Celgene, Summit, NJ, USA), an albumin-bound paclitaxel, is currently in clinical use for the treatment of breast cancer.[29, 30]

NK105 is a paclitaxel-incorporating “core-shell-type” polymeric micellar nanoparticle formulation.[31] Water-soluble and amphiphilic polymers (the shell), composed of a hydrophilic polyethylene glycol (PEG) and a hydrophobic polyaspartate, can form micelles with hydrophobic paclitaxel molecules enclosed in the micellar core. Thus, paclitaxel is made water-soluble without the need of Cremophor EL or ethanol. On average, each NK105 micelle measures approximately 85 nm in diameter, which is considered adequate to effectively utilize the EPR effect. Previous studies in animal cancer models have reported that intravenous administration of NK105 results in enhanced antitumor effects and reduced toxicity compared with PTX-Cre.[31, 32] The safety and pharmacokinetic advantages of NK105 in humans were demonstrated in a Phase I trial,[33] whereas a Phase II trial of NK105 for advanced gastric cancer with failure of first-line chemotherapy reported an overall response rate of 25% and median overall survival of 14.4 months.[34]

The intraperitoneal administration of nanoparticulate anticancer agents for the treatment of peritoneal dissemination has not been investigated extensively, despite the existence of data indicating the potency of this type of treatment.[35] Previously, we showed that intraperitoneal administration of nanoparticulate paclitaxel results in deeper penetration of the drug into peritoneal nodules and enhanced antitumor effects compared with PTX-Cre.[36, 37] In the present study, we evaluated the intraperitoneal and systemic antitumor effects of NK105 administered intraperitoneally in an animal model of mouse. Moreover, we evaluated, for the first time, the pharmacokinetics of nanoparticulate paclitaxel following intraperitoneal administration.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure Statement
  8. References

Materials

Both PTX-Cre and NK105 were supplied by Nippon Kayaku (Tokyo, Japan). The PTX-Cre was supplied as a liquid solution of paclitaxel in Cremophor EL and ethanol (1 : 1, v/v) at a concentration of 6 mg/mL paclitaxel. The NK105 was supplied as a powder formulation and contained 4.1% (w/w) paclitaxel, which had been prepared as reported previously.[31, 32] Briefly, polymeric micellar particles were formed by the self-association of amphiphilic block copolymers in an aqueous medium. The NK105 polymer was generated using PEG as the hydrophilic segment and modified polyaspartate as the hydrophobic segment. The carboxylic groups of the polyaspartate block were modified by an esterification reaction with 4-phenyl-1-butanol, resulting in the conversion of half of the groups to 4-phenyl-1-butanolate. The molecular weight of the polymers was determined to be approximately 20 000 (PEG block: 12 000; modified polyaspartate block: 8000). A self-association process was used to incorporate paclitaxel into the inner core of the micelle system by physical entrapment through hydrophobic interactions between the drug and specifically designed block copolymers for paclitaxel. This process resulted in the formation of NK105, a paclitaxel-incorporating polymeric micellar nanoparticle formulation with a single narrow size distribution. The weight-average diameter of the nanoparticles was approximately 85 nm and ranged from 20 to 430 nm. The drug formulation was diluted using PBS or normal saline.

Cell culture

The human gastric cancer variant line MKN45P, established from gastric cancer that disseminated to the peritoneal cavity,[38] was cultured routinely in DMEM (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% FBS (Sigma-Aldrich), 100 units/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich) at 37°C in a humidified atmosphere of 5% CO2 in air. After cells reached subconfluence, they were removed by treatment with a commercially available solution of 1 mM EDTA and an animal origin-free alternative for porcine trypsin (TrypLE Express; Invitrogen, Carlsbad, CA, USA) before being used in the experiments.

Animals

Specific-pathogen-free conditioned female BALB/c nude mice were purchased from Charles River Japan (Yokohama, Japan) and housed in an air-conditioned (23°C) room under a 12-h light–dark cycle. Female white Japanese rabbits weighing 3.0–3.5 kg were purchased from Saitama Rabbitry (Saitama, Japan), housed individually and allowed free access to food and water. All animal experiments were performed in accordance with the Guidelines for Animal Experiments of The University of Tokyo.

In vitro cell proliferation assay

The cytotoxic effects of PTX-Cre and NK105 on the in vitro growth of MKN45P cells were evaluated in a cell proliferation assay. Briefly, MKN45P cells (1 × 103 cells in 100 μL/well) were seeded into a 96-well microtiter flat-bottomed plate in DMEM containing 10% FBS. The cells were cultured overnight at 37°C in 5% CO2 to allow attachment. The medium was then aspirated and fresh medium containing 10% FBS and varying concentrations of PTX-Cre and NK105 (0.001, 0.01, 0.1, and 1 μg/mL paclitaxel) were added to three replicate wells. After incubation at 37°C in 5% CO2 for 72 h, the number of living cells was determined using an MTS assay (CellTiter 96 AQueous One Solution Cell Proliferation Assay; Promega, Madison, WI, USA) according to the manufacturer's instructions. The assay consisted of a tetrazolium compound (inner salt; 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium [MTS]) and an electron-coupling reagent, namely phenazine methosulfate (PMS). In this assay, the MTS is converted by dehydrogenase enzymes into a formazan product that is found in metabolically active cells and is soluble in tissue culture medium. The absorbance of formazan was measured directly at 490 nm in a 96-well plate using a microtiter plate reader (ThermoFisher, Waltham, MA, USA) and was directly proportional to the number of living cells in the culture.

Intraperitoneal administration of PTX-Cre or NK105 in a mouse model of subcutaneous tumors and peritoneal dissemination of gastric cancer cells

To evaluate systemic antitumor effects, 8-week-old female BALB/c nude mice were inoculated simultaneously with 2.0 × 106 MKN45P cells suspended in 1 mL PBS intraperitoneally and 1.0 × 106 MKN45P cells suspended in 200 μL PBS subcutaneously. The mice were randomly divided into three groups (control, PTX-Cre, and NK105). The control group consisted of 10 mice that were injected with PBS intraperitoneally; the PTX-cre and NK105 groups consisted of seven mice each that were treated with a dose of PTX-cre and NK105, respectively, that was equivalent to 40 mg/kg paclitaxel. The control group was not injected with a vehicle for PTX-Cre or NK105, but was injected with PBS because: (i) previous data have demonstrated that Cremophor EL, the vehicle for PTX-Cre, does not have an antitumor effect compared with PBS[11, 39]; and (ii) the amphiphilic polymers that constitute the outer shell of the NK105 micelle are not stable without paclitaxel as the inner core. The drugs were administered intraperitoneally on Days 7 and 14 after inoculation of MKN45P cells. The total volume of liquid injected was set to 1.0 mL to optimize spreading of the drugs throughout the entire peritoneal cavity. Body weight was monitored to evaluate toxic effects.

Caliper measurements of the longest (L) and shortest (S) diameters (mm) of subcutaneous tumors were performed three times a week. The formula for an ellipsoid sphere [(× S2)/2] was used to calculate tumor volume. On Day 19 after inoculation, mice were killed and dissected. Each mouse was anesthetized using diethyl ether inhalation and a whole blood sample was collected by transthoracic cardiac puncture; the mouse was then killed and a laparotomy performed. The number of peritoneal nodules that had grown >0.5 mm in diameter was counted and the total weight of the nodules was measured. Serum was separated from the blood samples by centrifugation at 700g for 5 min at 4°C. Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and urea nitrogen (UN) were then measured using the ultraviolet method; creatinine levels were determined using enzymatic methods in a commercial laboratory (SRL, Tokyo, Japan).

Measurement of paclitaxel concentrations in ascitic fluid, peritoneal nodules, and other organs after intraperitoneal administration of PTX-Cre or NK105 in mice

Seven-week-old female BALB/c nude mice were inoculated intraperitoneally with 3.0 × 106 MKN45P cells suspended in 1 mL PBS. On Day 14 after inoculation of MKN45P cells, mice were randomly divided into two groups of nine mice each. One group was injected intraperitoneally with PTX-Cre, whereas the other was injected with NK105. The concentration of paclitaxel administered in both groups was 40 mg/kg in a fixed volume of 1 mL. Three mice from each group were killed 4, 24, and 48 h after drug administration. Residual peritoneal fluid was carefully collected and its volume measured. The peritoneal nodules and liver were excised. Tissue samples were washed in saline and processed after drying the surface moisture. Tissue samples were accurately weighed and homogenized in 1 mL TBS containing 1% Tween 20 (Sigma-Aldrich). The homogenate was centrifuged at 11 270g for 5 min and the supernatant collected. The residual peritoneal liquid and the supernatant from the tissue samples were frozen at −20°C until analysis and determination of paclitaxel concentrations using reverse-phase HPLC by a commercial laboratory (SRL).

Measurement of serum paclitaxel concentrations after intraperitoneal administration of PTX-Cre or NK105 in rabbits

Rabbits were anesthetized with an intramuscular injection of a mixture of ketamine (50 mg/kg) and xylazine (3 mg/kg) and then underwent laparotomy through a small incision in the middle abdomen. An 8-Fr catheter was inserted through the abdominal incision for drug administration. Rabbits were randomly divided into two groups of three rabbits each. The rabbits in each group were administered a single slow bolus intraperitoneal injection of PTX-Cre or NK105 (5 mg/kg paclitaxel in 25 mL saline), after which the abdominal incision was closed. Peripheral blood was collected from each rabbit via an ear vein at 0.5, 6, 12, 24, and 48 h after drug administration. The serum was separated by centrifugation at 700g for 5 min at 4°C, and the concentration of paclitaxel was determined using reverse-phase HPLC. The area under the curve (AUC) was calculated using the trapezoidal rule.

Statistical analyses

For the MTS assay and blood sample analyses, results were evaluated statistically using Student's t-test; post hoc multiple comparison tests were performed by analysis of variance (anova) followed by the Tukey–Kramer method. These results are given as the mean ± SD. Data regarding the number, weight, and volume of subcutaneous and peritoneal tumors in mice were evaluated statistically using the Kruskal–Wallis' test, with post hoc multiple comparisons performed using Wilcoxon's rank-sum test with Bonferroni correction. For all experiments, < 0.05 was considered significant. All statistical analyses were performed using JMP version 9.0 (SAS Institute, Cary, NC, USA).

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure Statement
  8. References

In vitro cell proliferation assay

The in vitro proliferative activity of MKN45P cells was similarly dose-dependently decreased following the addition of PTX-Cre and NK105 (Fig. 1). There were no significant differences between PTX-Cre and NK105 at any dose tested on the proliferative activity of MKN45P cells.

image

Figure 1. In vitro cytotoxic effects of paclitaxel solubilized in Cremophor EL and ethanol (PTX-Cre) and the paclitaxel-incorporating micellar nanoparticle (NK105). MKN45P cells were seeded in 96-well plates overnight before exposure to each agent at concentrations ranging from 0.001 to 1 μg/mL. Cells were treated for a maximum of 72 h before 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) staining. There were no significant differences in cell viability between the two groups for any dose tested. Data are the mean ± SD of three different experiments.

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Antitumor effects of intraperitoneal NK105 on subcutaneous tumors and peritoneal dissemination of gastric cancer in mice

On Day 19 after inoculation of MKN45P cells, two mice in the control group died, whereas all mice in the remaining two groups were alive. Representative pictures of the tumors in the three groups shown in Figure 2. The volume and weight of subcutaneous tumors were significantly reduced in the NK105 group compared with the control group, whereas no significant reduction was observed in the PTX-Cre group (Fig. 3a,b). The number and weight of peritoneal nodules were significantly reduced in both the PTX-Cre and NK105 groups compared with the control group, with a significantly greater reduction seen in the NK105 group compared with PTX-Cre group (Fig. 3c,d). Table 1 lists body weight, which could be attributed to the tumor burden and toxic effect, and serum AST, ALT, UN, and creatinine levels at the time of death of mice in the three groups. There were no significant differences between the PTX-Cre and NK105 groups in any of the parameters evaluated.

image

Figure 2. Representative photographs of subcutaneous tumors and peritoneal nodules at the time of death in (a,d) the control group, (b,e) the group treated with paclitaxel solubilized in Cremophor EL and ethanol (PTX-Cre) and (c,f) the group treated with the paclitaxel-incorporating micellar nanoparticle (NK105).

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image

Figure 3. In vivo antitumor effects of paclitaxel solubilized in Cremophor EL and ethanol (PTX-Cre) and the paclitaxel-incorporating micellar nanoparticle (NK105). MKN45P cells were inoculated simultaneously both subcutaneously and intraperitoneally, and PTX-Cre or NK105 was administered intraperitoneally on Days 7 and 14 (arrows in [a]). (a) The volume of the subcutaneous tumor was calculated as an ellipsoid sphere and the median volume is shown for each group. At the time of death, tumor volume was significantly less in the NK105 group compared with the control group (**< 0.01), whereas no significant differences were observed between the control and PTX-Cre groups in either tumor volume (a) or weight (b). (c,d) Results regarding the number of peritoneal nodules >0.5 mm (c) and the weight of the peritoneal nodules (d) indicate that NK105 is more effective than PTX-Cre. The box and whisker plots show the minimum, 25th percentile, median, 75th percentile, and maximum values. *< 0.05, **< 0.01.

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Table 1. Body weight and liver and renal function at the time of death (on Day 19) in the control and two treatment groups
ControlPTX-CreNK105
  1. Data are the mean ± SD. There were no significant differences between the two treatment groups. PTX-Cre, paclitaxel solubilized in Cremophor EL and ethanol; NK105, paclitaxel-incorporating micellar nanoparticle; AST, aspartate aminotransferase; ALT, alanine aminotransferase; UN, urea nitrogen.

Body weight (g)18.1 ± 1.019.5 ± 1.619.9 ± 2.5
AST (IU/L)419 ± 156226 ± 127223 ± 149
ALT (IU/L)79 ± 3146 ± 1248 ± 12
UN (mg/dL)42 ± 546 ± 248 ± 3
Creatinine (mg/dL)0.19 ± 0.030.14 ± 0.020.14 ± 0.02

Tissue distribution of paclitaxel after intraperitoneal administration of NK105 in peritoneal tumor-bearing mice

The weight of the residual liquid after intraperitoneal administration of drugs and the paclitaxel concentration in the fluid were significantly higher in the PTX-Cre group at all time points evaluated (Fig. 4a,b). The paclitaxel concentration in the mesenteric peritoneal nodules was significantly higher in the NK105 group than the PTX-Cre group 4 h after intraperitoneal administration, whereas paclitaxel concentrations in the liver were similar between the two groups (Fig. 4c,d).

image

Figure 4. Mice were killed 4, 24, and 48 h after intraperitoneal administration of paclitaxel solubilized in Cremophor EL and ethanol (PTX-Cre) or the paclitaxel-incorporating micellar nanoparticle (NK105). (a) The weight of the residual liquid was significantly higher in the PTX-Cre group. Paclitaxel concentrations in residual peritoneal liquid (b), peritoneal nodules (c), and liver (d) were determined by reverse-phase HPLC. Paclitaxel concentrations were significantly higher in peritoneal nodules in the NK105 group than in the PTX-Cre group 4 h after administration, whereas there were no significant differences between the two groups in the liver. Data are the mean ± SD for three mice in each group at each time point. (Note, paclitaxel concentrations in residual peritoneal liquid in the NK105 group were not measurable at 48 h.) *< 0.05, **< 0.01, ***< 0.001.

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Serum paclitaxel concentrations after intraperitoneal administration of NK105 to rabbits

Serum paclitaxel concentrations after intraperitoneal administration of the drugs is shown in Figure 5. At 6, 12, 24, and 48 h after intraperitoneal administration of the drugs, serum paclitaxel concentrations in were significantly higher in the NK105 group than in the PTX-Cre group, whereas they were higher in the PTX-Cre group 0.5 h after intraperitoneal administration. The time of peak concentrations (T max) was 6 h in both groups. Both the peak concentration (C max) and the AUC0–48h were significantly higher in the NK105 group compared with the PTX-Cre group (Table 2).

image

Figure 5. Pharmacokinetics of intraperitoneally administered paclitaxel solubilized in Cremophor EL and ethanol (PTX-Cre) or the paclitaxel-incorporating micellar nanoparticle (NK105). Rabbits were injected intraperitoneally with PTX-Cre or NK105 (5 mg/kg paclitaxel) in 25 mL saline. Peripheral blood was collected via an ear vein at 0.5, 6, 12, 24, and 48 h after drug administration, and the concentration of paclitaxel determined by reverse-phase HPLC. Paclitaxel concentrations were significantly higher in the NK105 group than in the PTX-Cre group at 6, 12, and 24 h, but were significantly higher in the PTX-Cre group at 0.5 h. Data are the mean ± SD of three rabbits in each group. *< 0.05, **< 0.01, ***< 0.001.

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Table 2. Pharmacokinetic parameters of serum paclitaxel following intraperitoneal administration
PTX-CreNK105 P-value
  1. Data are the mean ± SD. PTX-Cre, paclitaxel solubilized in Cremophor EL and ethanol; NK105, paclitaxel-incorporating micellar nanoparticle; T max, time at which the maximum drug concentration was observed; C max, maximum drug concentration; AUC0–48 h, area under the blood concentration time curve from 0 to 48 h.

T max (h)6 ± 06 ± 01.000
C max (ng/mL)108 ± 2524 100 ± 3560<0.001
AUC0–48 h (ng·h/mL)1500 ± 108191 000 ± 32 100<0.001

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure Statement
  8. References

The primary aims of the present study were to clarify whether NK105 has any advantages over PTX-Cre in intraperitoneal chemotherapy for peritoneal dissemination and to examine the kinetics of intraperitoneally administered NK105. Treatment of mice with NK105 resulted in a significantly greater reduction in both peritoneal and subcutaneous tumors compared with PTX-Cre treatment. The pharmacokinetics study showed that intraperitoneally administered NK105 was absorbed rapidly into the systemic blood stream and that serum paclitaxel concentrations were maintained at significantly higher levels for longer in the NK105 compared with the PTX-Cre group.

Although the effects of intravenous NK105 have been reported by several studies,[31, 33] the effects of this nanoparticulate paclitaxel following intraperitoneal administration have not been investigated. An ideal agent for intraperitoneal chemotherapy should exit slowly from the peritoneal cavity to penetrate the peritoneal nodules from the surface.[15] Water-soluble, low-molecular weight agents such as 5-fluorouracil or cisplatin are inappropriate for intraperitoneal chemotherapy because they are rapidly absorbed via the capillary blood vessels of the peritoneum after intraperitoneal administration.[15]

We initially expected NK105 to be retained in the peritoneal cavity longer than PTX-Cre following intraperitoneal administration because of its larger particle size. However, NK105 disappeared from the peritoneal cavity more rapidly than PTX-Cre, resulting in increased serum concentrations of paclitaxel.

After absorption into the systemic blood stream, the Cmax and AUC values were significantly higher in the NK105 compared with PTX-Cre group, which is consistent with previously reported patterns after intravenous administration of NK105.[33] It is reasonable that the reduction of subcutaneous tumors was greater in the NK105 group, because high serum concentrations and longer drug retention may produce an enhanced systemic antitumor effect. This enhanced systemic circulation of NK105 and the EPR effect may have contributed to a considerable extent to the regional antitumor effect against peritoneal nodules.

However, 4 h after intraperitoneal administration, when the transfer of drugs to the systemic circulation was probably not yet complete, the concentration of paclitaxel in peritoneal nodules was significantly higher in the NK105 group than in the PTX-Cre group, even though paclitaxel concentrations in the liver were similar in the two groups. This high paclitaxel concentration in peritoneal nodules in the NK105 group, especially at an early time point after intraperitoneal administration, may be due to enhanced direct penetration of nanoparticulate paclitaxel from the surface of the nodules, which has been shown previously using a different nanomicellar paclitaxel formulation.[36, 37] Another PEG-conjugated nanomicellar paclitaxel exhibited enhanced tumor accumulation and deeper penetration in a model of disseminated ovarian cancer.[24] Thus, the route of administration (i.e. the direct penetration of the drug from the surface) may also have contributed to the enhanced antitumor effect of NK105 against peritoneal tumors.

The reasons why nanomicellar particles can penetrate deeply into the nodules or are absorbed from the peritoneum remain unclear, but an interesting affinity of cell membranes and micelles has been suggested. Certain amphiphilic polymers have been reported to penetrate non-endocytically or to fuse with plasma membranes and enter the cytoplasm of living cells within a few minutes without bilayer disruption.[40] One of the problems associated with intravenous chemotherapy is the high interstitial pressure of solid tumors, which prevents chemotherapeutic agents, including nanoparticles, from leaking from the vessels and penetrating into the tumor tissue.[41-44] Other possibilities explaining the rapid absorption may include, for example, effective penetration of the drug into the tumor interstitial tissue and enhanced uptake by the lymphatic stomata or milky spots of the peritoneum. However, further studies are necessary to evaluate how nanomicellar paclitaxel, including NK105, penetrates into the tumor tissue and peritoneum.

The pharmacokinetics of intravenously injected NK105 have been characterized in both a mouse model[31] and in humans.[33] Both studies indicated a prolonged presence of NK105 in the systemic circulation compared with PTX-Cre. This long systemic retention indicates that NK105 exists in the blood stream in micellar form because free paclitaxel would be metabolized earlier. Therefore, intraperitoneally administered NK105 may exist in micellar form after absorption into the blood stream, which prolongs its presence in the systemic circulation.

Another advantage of NK105 is its water solubility without the need for Cremophor EL and ethanol. In preliminary experiments, we administered PTX-Cre and NK105 intraperitoneally in escalating doses (data not shown). Intraperitoneal administration of 50 mg/kg PTX-Cre had a deleterious effect on mice, which shivered and otherwise failed to move for several hours. At a dose of 80 mg/kg PTX-Cre, the mice did not survive longer for than 1 day. The same effects were seen when mice were treated with the Cremophor and ethanol vehicle alone. However, no apparent acute toxic effect was observed in the NK105 group, even at doses of 100 mg/kg administered intraperitoneally. Although NK105 is clearly less toxic than PTX-Cre, we administered equal doses (40 mg/kg paclitaxel) to mice in the present study.

There were no significant differences in body weight loss, hepatic toxicity, or renal toxicity between the PTX-Cre and NK105 groups, although the serum concentrations of paclitaxel were higher in the NK105 group. Liver paclitaxel concentrations were similar in the two groups. The apparently lower systemic toxicity of NK105 despite the presence of higher serum concentrations may be due, in part, to the fact that macromolecules do not tend to leak from normal vessels (the EPR effect), but further studies are required to clarify this point.

Prolonged retention in the peritoneal cavity and rapid clearance from the systemic circulation are believed to be important attributes for ideal drugs designed for intraperitoneal chemotherapy.[45] Therefore, intravenous chemotherapy is generally combined with intraperitoneal chemotherapy to target primary sites or metastatic sites other than peritoneal dissemination. However, intraperitoneally administered NK105 may be sufficiently effective for both systemic and regional tumors because of its rapid transition from the peritoneum to the systemic circulation and its deep and rapid direct penetration into peritoneal nodules. This is a novel finding in the field of pharmacokinetics of intraperitoneal chemotherapy.

The present study has certain limitations. First, we were unable to use a single species of animal throughout all the experiments. Nude mice were used to evaluate the antitumor effect and different drug concentrations in peritoneal nodules, whereas rabbits were used for serum pharmacokinetic studies because of the need to obtain sufficient blood samples. Second, the effect of the polymer as the outer shell of NK105 alone was not examined in the present study. The amphiphilic polymer and paclitaxel form the outer shell and inner core of NK105, respectively; the paclitaxel in the inner core is essential for the stability of NK105. Thus, the polymeric micelle alone could not be used as a control for NK105. Third, hepatic and renal disorders were observed in the control group, which may have been the result of dehydration or cachexia due to the tumor burden itself. Therefore, further investigations may be required to more accurately evaluate the side effects of this type of therapy.

Intraperitoneally administered NK105 disappeared rapidly from the peritoneal cavity despite having a particle size larger than that of PTX-Cre. Future experiments should therefore be aimed at improving the efficacy of the system by prolonging the residence time of NK105 in the peritoneal cavity, with the expectation that this will increase drug penetration into the nodules. Controlling drug release and absorption by hydrogel conjugation,[11, 46] regulating peritoneal permeability by combination with bevacizumab,[47] molecular targeting or pH control release.[48-50] and other new strategies are promising methods that could be combined with intraperitoneal NK105.

In conclusion, intraperitoneal administration of NK105 was more effective in an animal model against gastric cancer with peritoneal dissemination than PTX-Cre due to its enhanced penetration into peritoneal nodules and higher and longer retention in the systemic circulation. Intraperitoneal chemotherapy using nanoparticulate paclitaxel could be a promising strategy for the treatment of gastric cancer with peritoneal dissemination. A clinical study of intraperitoneally administered NK105 is expected to be performed in the near future to evaluate its efficacy in gastric cancer patients.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure Statement
  8. References

The authors thank Chieko Uchikawa for her excellent technical assistance. This study was funded by the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Ministry of Health, Labour and Welfare of Japan.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure Statement
  8. References