SEARCH

SEARCH BY CITATION

Abstract

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

Anthracyclines have long been considered to be among the most active agents clinically available for the treatment of breast cancer despite their toxicity. To improve their pharmacological profiles, a new macromolecular prodrug, denoted NC-6300, was synthesized. NC-6300 comprises epirubicin covalently bound to polyethylene-glycol polyaspartate block copolymer through an acid-labile hydrazone bond. The conjugate forms a micellar structure spontaneously in aqueous media with a diameter of 60–70 nm. The block copolymers are partially substituted with hydrophobic benzyl groups to stabilize the micellar structure. The present study was designed to confirm that polymeric micelles incorporating epirubicin through an acid-labile linker improve the therapeutic index and achieve a broad range of therapeutic doses. Pharmacokinetic studies in rats showed highly enhanced plasma retention of NC-6300 compared with native epirubicin. The maximal tolerated doses in mice of NC-6300 and native epirubicin were 25 and 9 mg/kg, respectively, when administered three times with a 4-day interval between each dose. NC-6300 at 15 and 20 mg/kg with the same administration schedule regressed a Hep3B human hepatic tumor with slight and transient bodyweight loss. Remarkably, NC-6300 also inhibited growth of an MDA-MB-231 human breast tumor at the same dosage. In contrast, native epirubicin at 7 mg/kg administered three times with a 4-day interval was only able to slow tumor growth. Tissue distribution studies of NC-6300 showed efficient free epirubicin released in the tumor at 74% by area under the concentration-time curve (AUC) evaluation, supporting the effectiveness of NC-6300. In conclusion, NC-6300 improved the potency of epirubicin, demonstrating the advantage of NC-6300 attributable to the efficient drug release in the tumor. (Cancer Sci 2011; 102: 192–199)

Anthracyclines were first introduced for the treatment of metastatic breast cancer in the 1970s and are still among the most active single agents for the treatment of this disease despite their cardiotoxicity.(1) The aim of increasing their efficacy was first addressed using liposomes.(2) Efforts to design liposomes that are pH-sensitive, temperature-sensitive or antibody-targeted have all been pursued with various degrees of success.(3) However, current clinically approved liposomal formulations have still resulted in only modest increased efficacy for the treatment of cancer.(4) Its actual advantage is reduced toxicity rather than increased therapeutic effect.(5–7)

To increase the efficacy, polymer-based anthracyclines have also been studied extensively.(8,9) Recent strategies have been developed and successfully applied to attain desirable tumor localization through polymeric micelles composed of polyethylene glycol (PEG)-poly (amino acid) block copolymers.(10) These types of strategies involve drug release inside endosomes and lysosomes after cellular internalization, where the slightly acidic pH leads to cleavage of the acid-sensitive linkage.(11)

We have designed NC-6300 as a macromolecular prodrug, which can form a micellar structure spontaneously in aqueous media, with anthracycline conjugated inside the micelles. Epirubicin (EPI), the 4′-epimer of doxorubicin (DOX), was chosen as the active ingredient, because of its lower cardiotoxicity and equal efficacy compared with an equimolar DOX concentration.(12) Nevertheless, DOX-based formulations were also synthesized and studied (data not shown). For the NC-6300 preparation, PEG-polyaspartate block copolymers partially substituted with hydrophobic groups, such as the benzyl group, to obtain an advanced blood circulation time were used. This modification stabilized the micellar structure, increased plasma retention, and improved the therapeutic effect.(13)

The present study was designed to confirm drug release in the tumor in vivo after intravenous administration of NC-6300 for the first time and investigate its efficacy against triple-negative breast and liver tumors actively investigated clinically.(14–16) The plasma pharmacokinetics, tissue distribution and preliminary toxic profiles of NC-6300 were also investigated. This is because only the released drug has biological activity, and thus it is important to correlate the efficacy and toxicity with the levels of released EPI as a function of time. These results support enhanced efficacy and the possibility of reduction in cardiotoxicity of NC-6300.

Materials and Methods

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

Chemicals and reagents.  NC-6300 was synthesized at NanoCarrier Co., Ltd (Kashiwa, Japan). The structure is presented in Figure 1. The synthesis has been described elsewhere.(13) All of the chemicals were reagent-grade products obtained commercially.

image

Figure 1.  Chemical structure of NC-6300. Weight-average molecular weight of PEG is 12 kDa, corresponding to the value of m = 272. The number of Asp residue is 40, corresponding to the sum of n, o, p and q = 40. Each NC-6300 contains approximately eight epirubicin (EPI) molecules and 20 Bn groups, corresponding to the value of P = 8 and n = 20, respectively.

Download figure to PowerPoint

Cells and animals.  PC-3 human prostate tumor was purchased from American Type Culture Collection through Summit Pharmaceuticals International (Tokyo, Japan). MDA-MB-231 human breast and Hep3B human hepatic tumors were obtained from European Collection of Cell Cultures through DS Pharma Biomedical (Osaka, Japan). Male Wistar rats, male BALB/c mice, and female nude mice (BALB/c nu/nu) were purchased from Charles River Japan (Yokohama, Japan). Male nude mice (BALB/c nu/nu) were from CLEA Japan (Tokyo, Japan).

Micelle preparation.  NC-6300 was first dissolved in purified water to allow spontaneous formation of micelles (2 mg of EPI/mL). The micelles were passed through a Millipore 0.22-μm filter (Millex GP PES Express; Billerica, MA, USA) and were frozen at −80°C after the addition of final 10% (w/v) sucrose until use. Their particle size was measured by a Malvern Zetasizer 3000HSA (Malvern, Worcestershire, UK) at 25°C. The average value was 60–70 nm.

Release study of EPI from NC-6300.  NC-6300 (50 μL) was added into 0.1 M of various buffers (950 μL) prewarmed at 37°C with a final EPI concentration of 10 μg/mL, including sodium phosphate (pH 7.4, 7.0 and 6.0), sodium acetate (pH 5.0 and 4.0) and ammonium formate (pH 3.0) buffers. The mixture was kept at 37°C. At given time intervals, aliquots of the samples (50 μL) were taken and compensated with the same buffer. The released EPI was measured by HPLC.

In vitro cytotoxicity.  PC-3 and MDA-MB-231 cells were maintained in RPMI1640 media supplemented with 10% heat-inactivated fetal bovine serum (FCS) at 37°C under an atmosphere of 5% CO2. Exponentially growing cells were typically seeded in 96-well plates (5000 cells/well) and cultured in the media overnight. The cells were then treated with either NC-6300 or native EPI for 72 h. A WST-8 reagent (Dojindo Laboratories, Kumamoto, Japan) was added to each well and cell viability was calculated according to a manual from the manufacturer. The drug concentration that was required to inhibit cell growth by 50% (GI50) was determined from the dose-response curves.

Toxicity dose-finding studies.  Healthy male BALB/c mice and male nude mice xenografted with PC-3 prostate tumor were used for toxicity dose-finding studies. Mice received intravenous administration of either NC-6300 or native EPI three times with a 4-day interval at a dose ranging 7–12 mg/kg for EPI and 15–40 mg/kg for NC-6300, respectively. A dose of NC-6300 is hereafter always expressed as EPI equivalent mg/kg of bodyweight per injection. Mice were observed for 28 days after the first injection. The bodyweight of animals was monitored two or three times a week. The maximum tolerable dose (MTD) was defined as the maximum dose that caused no drug-related lethality and that induced animal bodyweight loss of <20% of original weight.

Pharmacokinetic study in rats.  Healthy male Wistar rats received a single bolus intravenous injection of either NC-6300 or EPI at 1 mg of EPI/kg (n = 3). Immediately before blood sampling, rats were anesthetized with ether and blood samples were collected at 0.083, 1, 2, 4, 6, 8 and 24 h for NC-6300 and at 2, 5, 10, 15, 30 and 60 min for native EPI, respectively. Plasma was harvested by centrifugation at 4°C and stored at −30°C until analyzed.

Tissue distribution of NC-6300 in tumor-bearing mice.  MDA-MB-231 cells were suspended in a 50% Matrigel (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and inoculated subcutaneously to the back of female 5-week-old BALB/c nu/nu mice. When tumor volumes reached approximately 300 mm3, the mice received an intravenous injection of either native EPI (7 mg/kg) or NC-6300 (20 mg/kg). For each of the drugs, 24 mice were divided into eight groups (= 3), corresponding to 0.083, 1, 6, 24, 48, 72, 120 and 168 h. Immediately before sampling, the mice were anesthetized with ether and blood was collected from the heart using a heparinized syringe. Plasma was harvested by centrifugation at 4°C. The tumor, liver, spleen, kidney, heart and lung were removed, rinsed with saline and stored at −30°C until analyzed.

Determination of drug concentration in plasma and tissue.  Tissue samples were suspended in 0.1 M sodium phosphate buffer (pH 7.4) at a concentration of 25% w/w and homogenized on ice using a Polytron PT3100 mixer (Kinematica, Lucerne, Switzerland). Using aliquots of the homogenates and plasma (50 μL), both the released EPI (that released in vivo from NC-6300) and the total EPI (that released from NC-6300 plus the remainder of NC-6300) concentrations were determined by HPLC.

To determine the released EPI concentration, the homogenates and plasma samples (50 μL) were treated with acetonitrile (125 μL) to precipitate proteins. One percentage of Triton X-100 (25 μL) was added and the sample was vortexed and centrifuged for 5 min at 5000g at 4°C. Daunorubicin HCl (2 μg/mL)/20 mM sodium phosphate buffer (pH 7.4, 75 μL) was added to the clear supernatant (75 μL) as an internal standard. The prepared mixture was analyzed by HPLC.

To determine the total EPI concentration, the samples (50 μL) were treated with acetonitrile (130 μL) and acidified with 1 N HCl (20 μL) for 1 h at room temperature to permit the complete release of EPI from NC-6300. Similarly, 1% of Triton X-100 (25 μL) was added and the sample was vortexed and centrifuged for 5 min at 5000g at 4°C. Daunorubicin HCl (2 μg/mL)/25 mM ammonium formate buffer (pH 3.0, 75 μL) was added to the clear supernatant (75 μL). The prepared mixture was analyzed by HPLC.

HPLC chromatography.  Reversed-phase HPLC was performed at 40°C on a Tosoh TSK-gel ODS-80TM (4.6ϕ × 150 mm) with a Tosoh ODS-80TM guard cartridge (Tokyo, Japan). The EPI was eluted with 25 mM ammonium formate buffer (pH3): acetonitrile (70:30, v/v) using a Waters Alliance System at a flow rate of 1.0 mL/min. Detection was carried out using a Waters fluorescence detector with excitation and emission wavelengths of 488 and 560 nm, respectively.

Preliminary toxicity studies.  Myelotoxicity and hepatotoxicity were assessed with male mice (Crlj:CD1 (ICR), = 10) intravenously administered three times with a 4-day interval with NC-6300 at either 15 or 20 mg/kg. General conditions and the bodyweight of animals were observed continuously for 49 days after the first administration. One animal per group was killed continuously for blood collection and autopsy (macroscopic observation of liver and bone marrow). Hematology and blood biochemistry tests were carried out using an ADVIA 120 automated hematology analyzer (Siemens Healthcare Diagnostics, Tokyo, Japan) and a 7180 clinical analyzer (Hitachi High-Technologies, Tokyo, Japan), respectively.

Evaluation of anti-tumor activity in human tumor xenograft models.

Human hepatic tumor Hep3B model.  Hep3B cells were grown in Eagle MEM medium supplemented with 10% FCS at 37°C under an atmosphere of 5% CO2. The cells were suspended in saline at 3 × 107 cells/mL and the cell suspension (0.1 mL) was inoculated in the back of male 7-week-old BALB/c nu/nu mice. Tumor growth was followed by measurements of tumor diameters with a sliding caliper two or three times a week. The tumor volume (TV) was calculated according to the following formula: TV = L × W2/2, where L and W are major and minor dimensions, respectively. Drug treatment with eight mice/group was started on day 13 after inoculation when the tumor volume reached 80 ± 34 mm3 (average ± SD). The EPI at 7 mg/kg and NC-6300 at 15 or 20 mg/kg were given by tail vein injection three times with a 4-day interval. The group of control mice was untreated. Efficacy was assessed as relative tumor volume to the original after the first administration. On day 28 the tumor was removed and the tumor growth inhibition rate (IR) was calculated using the following equation: IR (%) = (1 − T/C) × 100, where T and C are the tumor weights of the treated and control groups, respectively.

Human breast tumor MDA-MB-231 model.  Female tumor-bearing mice were used. Drug treatment with eight mice/group started on day 27 after inoculation when the tumor volume was 284 ± 60 mm3 (average ± SD). The EPI at 7 mg/kg and NC-6300 at 15 or 20 mg/kg were given by tail vein injection three times with a 4-day interval. The control mice group was untreated. Efficacy was similarly monitored.

Results

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

Drug release in vitro. In vitro drug release was investigated in a 100 mM buffer with various pH values. The results are shown in Figure 2, indicating pH-dependent drug release. At pH 3, approximately 80% of the EPI was released within 1 h, while 20% of the EPI release occurred at neutral pH. This result confirmed acid-sensitive EPI release from NC-6300.

image

Figure 2.  Epirubicin (EPI) release from NC-6300 in buffers with various pH values at 37°C. NC-6300 (50 μL) was added into 0.1 M of various buffers (950 μL) at 37°C with a final EPI concentration of 10 μg/mL. At given time intervals, aliquots of the samples (50 μL) were taken and released EPI was measured by HPLC.

Download figure to PowerPoint

In vitro cytotoxicity. In vitro cytotoxicity was examined using PC-3 and MDA-MB-231 tumor cells. It was found that the GI50s (equivalent to EPI) of NC-6300 against MDA-MB-231 and PC-3 were 0.7 and 1 μg/mL, while the values of EPI were 0.2 and 0.4 μg/mL, respectively. This is probably due to the limited drug release from NC-6300. Nevertheless, it should be noted that the released EPI was pharmacologically active.

Toxicity dose-finding studies.  To compare the anti-tumor effects of NC-6300 with EPI at their optimal doses, the MTD were first obtained in mice. In healthy mice, both NC-6300 at 30 mg/kg and EPI at 12 mg/kg three times with a 4-day interval caused a lethal effect and 20% bodyweight loss. Similar results were obtained in nude mice bearing PC-3. It was found that the MTD of NC-6300 and EPI were 25 and 9 mg/kg, respectively, at a schedule of three times with a 4-day interval.

Pharmacokinetic study in rats.  To investigate the conjugation effect on plasma pharmacokinetics, either EPI or NC-6300 was administered intravenously in rats at 1 mg of EPI/kg. Figure 3 compares the plasma concentration-time profile of total EPI after intravenous administration of NC-6300 with that of native EPI after intravenous injection of EPI itself. It was clearly shown that PEG-polyaspartate polymer gives EPI an extremely long-term circulation. When NC-6300 was administered, it continued to circulate at a high concentration for an extended period in the bloodstream. In contrast, EPI was cleared rapidly from the blood stream. Pharmacokinetic parameters were summarized in Table 1. The plasma concentration-time profiles of NC-6300 and EPI were well described by monoexponential and biexponential equations, respectively. The plasma area under the concentration-time curve (AUC) was calculated with extrapolation to infinity using the fitted model. NC-6300 increased the AUC approximately 2200-fold compared with EPI.

image

Figure 3.  Plasma concentration-time profiles of total epirubicin (EPI) after intravenous administration of NC-6300 (•) and EPI after injection of EPI itself (Δ) to rats at 1 mg of EPI/kg. Data points are the means of three rats. Bars (SD), if not shown, are within the size of the symbols.

Download figure to PowerPoint

Table 1.   Pharmacokinetic parameters of NC-6300 and epirubicin (EPI) after intravenous administration to rats at 1 mg of EPI/kg
DrugTerminal half-life (h)Vss (mL/kg)MRT (h)Cltotal (mL/kg per h)AUC0–∞ (μg h/mL)
  1. Parameters of NC-6300 and EPI were determined based on a one- and two-compartment model, respectively.

NC-63003.9949.45.768.57116.7
EPI0.4531410.1718 9610.053

Tissue distribution study in tumor-bearing mice.  Tissue distribution studies were performed to correlate the toxicity and efficacy results obtained for both NC-6300 and EPI with plasma and tissue drug concentrations. Nude mice xenografted with human breast tumor MDA-MB-231 received single intravenous injections of either NC-6300 at 20 mg/kg or EPI at 7 mg/kg, because this dose of each drug showed similar toxicity. Figure 4 shows the plasma concentration-time profiles of total EPI and released EPI after injection of NC-6300. These profiles in several tissues are summarized in Figure 5. Released EPI was observed in the blood circulation and various tissues. High levels of released EPI particularly were observed in the liver. The profiles of released EPI in all tissues measured reached their peak after 24 h. On the other hand, total EPI concentrations in the kidney, liver, spleen, lung and heart declined slowly with time. In contrast, the concentration in the tumor peaked around 24 h and subsequently declined very slowly, suggesting cellular uptake and retention of NC-6300. For comparison, profiles of EPI after administration of EPI itself are shown in Figures 4 and 5. The EPI was cleared rapidly from the body. At 72 h after injection, no retention of EPI was detected in the plasma and tumor.

image

Figure 4.  Plasma concentration-time profiles of total (•) and released epirubicin (EPI) (□) after intravenous administration of NC-6300 to nude mice bearing MDA-MB-231 breast tumor at 20 mg of EPI/kg. The profile of EPI after EPI injection at 7 mg/kg (Δ) is also shown for comparison. Data points are the means of three mice. Bars (SD), if not shown, are within the size of the symbols.

Download figure to PowerPoint

image

Figure 5.  Tissue concentration-time profiles of total (•) and released epirubicin (EPI) (□) after intra-venous administration of NC-6300 to nude mice bearing MDA-MB-231 breast tumor at 20 mg of EPI/kg. Profiles of EPI after EPI injection at 7 mg/kg (Δ) are also shown for comparison. Data points are the means of three mice. Bars (SD), if not shown, are within the size of the symbols.

Download figure to PowerPoint

To compare tissue accumulation of released EPI from NC-6300 with those of EPI after injection of native EPI, the AUC values in each tissue are summarized in Table 2. The ratio of released EPI to total EPI after administration of NC-6300 assessed by AUC for each tissue is also compared. NC-6300 increased the relative presence of EPI particularly in the plasma, tumor and liver, whereas NC-6300 decreased it in the kidney, lung and heart. The AUC value of released EPI in the tumor was 4.3-fold higher than that for the EPI solution at the same dose. In contrast, the AUC value of released EPI in the heart was only 0.28-fold compared with the native EPI solution. From these results, the therapeutic index of NC-6300 assessed by the AUC in the tumor and heart was estimated to be 15-fold (4.3/0.28) higher compared with the EPI solution. The release ratio was 20–46% in tissues; however, it was extremely high, 74%, in the tumor, suggesting an effective release of EPI from NC-6300 in the tumor.

Table 2.   AUC values in tissues after administration of either NC-6300 at 20 mg/kg or epirubicin (EPI) at 7 mg/kg in mice xenografted with MDA-MB-231 human breast tumor
 AUC/Dose (μg/g tissue h/mg/kg bodyweight)
NC-6300Native EPI
Released EPITotal EPI% Released†EPIRatio‡
  1. The AUC values of released, total, and native EPIs in tissues and plasma were calculated using the linear trapezoidal rule. †AUC of Released EPI/AUC of Total EPI × 100. ‡(AUC/Dose) of Released EPI after NC-6300 administration/(AUC/Dose) after EPI administration.

Tumor10.414.074.12.44.3
Spleen29.0146.519.822.41.3
Kidney24.553.445.846.70.52
Liver100.2230.443.518.85.3
Lung8.625.933.417.60.49
Heart2.07.527.07.10.28
Plasma9.7113.88.50.2145.9

Preliminary toxicity studies.  Toxicity preliminary focused on myelotoxicity and hepatotoxicity at near MTD was investigated. Figure 6 shows similar changes in the blood parameters after NC-6300 or EPI administration, indicating that the effect of conjugation of EPI on myelotoxicity is minimal. Figure 7 shows changes in hepatic parameters. It was found that NC-6300 increased slightly hepatic parameters such as AST, ALT and LD after the third administration (10–28 days after the first administration), followed by recovery to the normal level. Although high hepatic concentrations of EPI were observed after NC-6300 administration (Fig. 5), these results confirmed that NC-6300 shows no serious impact on the toxicity profile with transient and slight hepatotoxicity.

image

Figure 6.  Changes in the numbers of platelets (PLT), reticulocytes (Retic) and white blood cells (WBC). Drug injection three times with a 4-day interval (q4dx3) started on day 0. One mouse from each group was killed continuously for blood collection.

Download figure to PowerPoint

image

Figure 7.  Changes in the activity of aspartate transaminase (AST), alanine transaminase (ALT) and lactate dehydrogenase (LDH). Drug injection three times with a 4-day interval (q4dx3) started on day 0. One mouse from each group was killed continuously for blood collection.

Download figure to PowerPoint

Anti-tumor activity in human tumor xenograft models.  The in vivo efficacy of NC-6300 was evaluated against Hep3B hepatic tumor. Figure 8 shows the tumor growth and bodyweight change curves of nude mice given NC-6300 at 15 or 20 mg/kg three times with a 4-day interval, as compared with controls or animals given EPI at 7 mg/kg with the same schedule. NC-6300 regressed the tumor dose-dependently with a slight and transient bodyweight loss. In contrast, EPI reduced tumor growth to half, while it caused a >10% bodyweight loss. Tumor growth IR were calculated based on the tumor weight on day 28. The IR values for NC-6300 were 94.8% (15 mg/kg) and 99.3% (20 mg/kg), while it was 53.3% for EPI at 7 mg/kg (Table 3). These results demonstrate the superiority of NC-6300 over EPI.

image

Figure 8. In vivo efficacy of NC-6300 and epirubicin (EPI) against Hep3B human hepatic tumor ([a] tumor growth; [b] bodyweight change). Hep3B tumor cells (3 × 106 cells/animal) were inoculated subcutaneously on the back (day –13), and intravenous administration three times with a 4-day interval (q4dx3, indicated by the arrows) started on day 0 when the tumor volumes were 80 ± 34 mm3. The dose of NC-6300 is expressed as a dose equivalent to EPI. Each point represents the mean for eight mice. Bars represent the SD.

Download figure to PowerPoint

Table 3.   Tumor weight and inhibition rate on day 28 after the first administration of either NC-6300 or epirubicin (EPI) to nude mice xenografted with Hep3B tumor
GroupDose (mg/kg per injection)Tumor weight (g)IR (%)
  1. Each value of the inhibition rate (IR) represents the mean ± SD. *P < 0.001 vs the control group (Dunnett’s multiple comparison test). **P < 0.01 vs the control group (Student’s t-test). ***P < 0.001 vs the EPI group (Dunnett’s multiple comparison test).

Control1.88 ± 0.67
NC-6300150.098 ± 0.066*, ***94.8
NC-6300200.014 ± 0.010*, ***99.3
EPI70.88 ± 0.35**53.3

Additionally, the efficacy of NC-6300 was assessed against MDA-MB-231 breast tumor. As shown in Figure 9, NC-6300 exhibited remarkable cell growth inhibition with transient and slight bodyweight loss. In contrast, EPI at 7 mg/kg was only able to slow tumor growth.

image

Figure 9. In vivo efficacy of NC-6300 and epirubicin (EPI) against MDA-MB-231 human breast tumor ([a] tumor growth; [b] bodyweight change). MDA-MB-231 tumor cells (3 × 106 cells/animal) were inoculated subcutaneously on the back (day –27), and intravenous administration three times with a 4-day interval (q4dx3, indicated by the arrows) started on day 0 when the tumor volumes were 284 ± 60 mm3. The dose of NC-6300 is expressed as a dose equivalent to EPI. Each point represents the mean for eight mice. Bars represent the SD. Control versus EPI and EPI versus NC-6300 (15 mg/kg) were compared statistically in terms of tumor growth on days 21, 28, 35 and 42. * and ** indicate P < 0.05 and P < 0.01 (Student’s t-test), respectively.

Download figure to PowerPoint

Discussion

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

NC-6300 was designed to stay in the blood circulation for a long time and accumulate in the tumor based on the enhanced permeability and retention effect.(17) Superior anti-tumor activity is expected due to efficient drug release in the tumor after entry of NC-6300 into tumor cells, accompanied by exposure to an acidic environment of endosomes and/or lysosomes.(18,19) The present series of studies were performed to demonstrate the proof of this concept.

The above results supported the concept presented herein. Pharmacokinetic studies have confirmed that NC-6300 stays in the blood circulation for a long time at a high concentration. An in vitro drug release test clarified that an acidic environment accelerates drug release from NC-6300. A tissue distribution study indicated efficient drug release in the tumor. Efficacy studies showed superior anti-tumor activity of NC-6300 to that of EPI.

The superior effectiveness of NC-6300 might be responsible for efficient drug release in the tumor. Recently, released (bioavailable) DOX levels in the tumor after injection of DOX liposomes have been determined in tumor-bearing mice. It was reported that 27–49% of the liposomal DOX was bioavailable.(20) Compared with these values, although the drug is EPI instead of DOX, NC-6300 produced higher bioavailable EPI (74%) in the tumor (Table 2). Additionally, released EPI levels in the tumor after administration of NC-6300 at 20 mg/kg continued to be over the in vitro GI50 value for at least 1 week (Fig. 5), supporting the effectiveness of NC-6300.

The significant contribution of released EPI from NC-6300 to efficacy in vivo is supported by comparison of conjugates with a different linkage. Previously we have synthesized a similar DOX conjugate with an amide bond.(13) Although the conjugate stayed in the blood circulation for a long time after intravenous administration to rats, the conjugate was found to be ineffective. This is attributable to the stable amide bond, allowing very limited drug release in vivo, compared with NC-6300.

The rationale design of the linker for DOX-polymer conjugates has been studied extensively.(21,22) Retrospectively, DOX was covalently linked to poly-L-aspartic acid with a molecular weight of 20 000 through ester linkage and the preclinical efficacy was evaluated.(23) The conjugate was found to show a marginal therapeutic advantage over native DOX. Additionally, DOX was conjugated to PEG through amino linkage with a peptide linker.(24) The linker was designed to release DOX in lysosomes through the action of lysosomal enzymes. It was suggested these conjugates exhibit modest anti-tumor activity in correlation with the highest rate of DOX release in the presence of lysosomal enzymes. Currently, a growing number of research support the rationale for the selection of acid-sensitive hydrazone linkage for DOX-polymer conjugates in terms of improved efficacy.(25–28)

In terms of toxicity, NC-6300 increased hepatic concentrations (Fig. 5), thereby causing concern about hepatotoxicity. Changes in hepatic parameters in mice given NC-6300 at 20 mg/kg could be considered minimal and transient (Fig. 7). Compared with native EPI at 7 mg/kg, NC-6300 at 20 mg/kg produced higher hepatic levels of released EPI for a long time, probably leading to the changes. Additionally, our preliminary histopathological observation has confirmed vasculazation of Kupffer cells in the liver of mice receiving empty micelles or NC-6300 (data not shown), suggesting the need for attention to impairment of phagocytic activity, similar to liposomal DOX.(29,30)

In summary, NC-6300 displayed superior anti-tumor activity to that of EPI. Altered pharmacokinetic properties of NC-6300 in terms of plasma half-life, tumor accumulation and drug release might account for the effectiveness. Results presented here demonstrate proof of the concept that polymeric micelles with EPI covalently bound to the block copolymer through a hydrazone linkage improve the therapeutic index and achieve a broad range of therapeutic doses.

Acknowledgments

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

The authors thank Tsubasa Kondo, Tomomi Chijiiwa and Masami Tsuchiya for their technical assistance.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  • 1
    Swain SM, Whaley FS, Ewer MS. Congestive heart failure in patients treated with doxorubicin: a retrospective analysis of three trials. Cancer 2003; 97: 286979.
  • 2
    Perez-Soler R, Priebe W. Anthracycline antibiotics with high liposome entrapment: structural features and biological activity. Cancer Res 1990; 50: 42606.
  • 3
    Dass CR, Walker TL, Burton MA, Decruz EE. Enhanced anticancer therapy mediated by specialized liposomes. J Pharm Pharmacol 1997; 49: 9725.
  • 4
    Gracia AA, Kempf RA, Rogers M, Muggia FM. A phase II study of Doxil (liposomal doxorubicin): lack of activity in poor prognosis soft tissue sarcomas. Ann Oncol 1998; 9: 11313.
  • 5
    Safra T, Muggia F, Jeffers S et al. Pegylated liposomal doxorubicin (Doxil): reduced clinical cardiotoxicity in patients reaching or exceeding cumulative doses of 500 mg/m2. Ann Oncol 2000; 11: 102933.
  • 6
    O’Brien MER, Wigler N, Inbar M et al. Reduced cardiotoxicity and comparable efficacy in a phase III trial of pegylated liposomal doxorubicin HCl (CAELYX™/Doxil®) versus conventional doxorubicin for first-line treatment of metastatic breast cancer. Ann Oncol 2004; 15: 4409.
  • 7
    Theodoulou M, Hudis C. Cardiac profiles of liposomal anthracyclines. Greater cardiac safety versus conventional doxorubicin? Cancer 2004; 100: 205263.
  • 8
    Seymour LW, Ferry DR, Kerr DJ et al. Phase II studies of polymer-doxorubicin (PK1, FCE28068) in the treatment of breast, lung and colorectal cancer. Int J Oncol 2009; 34: 162936.
  • 9
    Duncan R, Vicent MJ, Greco F, Nicholson RI. Polymer-drug conjugates: towards a novel approach for the treatment of endrocine-related cancer. Endocr Relat Cancer 2005; 12: S18999.
  • 10
    Matsumura Y, Kataoka K. Preclinical and clinical studies of anticancer agent-incorporating polymer micelles. Cancer Sci 2009; 100: 5729.
  • 11
    Bae Y, Nishiyama N, Fukushima S, Koyama H, Matsumura Y, Kataoka K. Preparation and biological characterization of polymeric micelle drug carriers with intracellular pH-triggered drug release property: tumor permeability, controlled subcellular drug distribution, and enhanced in vivo antitumor efficacy. Bioconjug Chem 2005; 16: 12230.
  • 12
    Glück S. The expanding role of epirubicin in the treatment of breast cancer. Cancer Control 2002; 9: 1627.
  • 13
    Bobe I, Shibata N, Saito H, Harada M. Block copolymer for drug complex and pharmaceutical composition. PCT Patent Application WO 2008/047948.
  • 14
    Jaspers JE, Rottenberg S, Jonkers J. Therapeutic options for triple-negative breast cancers with defective homologous recombination. Biochim Biophys Acta 2009; 1796: 26680.
  • 15
    Simonetti RG, Liberati A, Angiolini C, Pagliaro L. Treatment of hepatocellular carcinoma: a systematic review of randomized controlled trials. Ann Oncol 1997; 8: 11736.
  • 16
    Epstein RJ, Leung TW. Reversing hepatocellular carcinoma progression by using networked biological therapies. Clin Cancer Res 2007; 13: 117.
  • 17
    Maeda H, Wu J, Sawa T, Matsumura Y, Hori K. Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J Control Release 2000; 65: 27184.
  • 18
    Charrois GJR, Allen TM. Drug release rate influences the pharmacokinetics, biodistribution, therapeutic activity, and toxicity of pegylated liposomal doxorubicin formulations in murine breast cancer. Biochim Biophys Acta 2004; 1663: 16777.
  • 19
    Kong G, Anyarambhatla G, Petros WP et al. Efficacy of liposomes and hyperthermia in a human tumor xenograft model: importance of triggered drug release. Cancer Res 2000; 60: 69507.
  • 20
    Laginha KM, Verwoert S, Charrois GJR, Allen TM. Determination of doxorubicin levels in whole tumor and tumor nuclei in murine breast cancer tumors. Clin Cancer Res 2005; 11: 69449.
  • 21
    Duncan R. Designing polymer conjugates as lysosomotropic nanomedicines. Biochem Soc Trans 2007; 35: 5660.
  • 22
    Hovorka O, Etrych T, Subr V, Strohalm J, Ulbrich K, Ríhová B. HPMA based macromolecular therapeutics: internalization, intracellular pathway and cell death depend on the character of covalent bond between the drug and the peptidic spacer and also on spacer composition. J Drug Target 2006; 14: 391403.
  • 23
    Pratesi G, Savi G, Pezzoni G et al. Poly-L-aspartic acid as a carrier for doxorubicin: a comparative in vivo study of free and polymer-bound drug. Br J Cancer 1985; 52: 8418.
  • 24
    Veronese FM, Schiavon O, Pasut G et al. PEG-doxorubicin conjugates: influence of polymer structure on drug release, in vitro cytotoxicity, biodistribution, and antitumor activity. Bioconjug Chem 2005; 16: 77584.
  • 25
    Bae Y, Kataoka K. Intelligent polymeric micelles from functional poly(ethylene glycol)-poly(amino acid) block copolymers. Adv Drug Deliv Rev 2009; 61: 76884.
  • 26
    MacKay JA, Chen M, McDaniel JR, Liu W, Simnick AJ, Chilkoti A. Self-assembling chimeric polypeptide-doxorubicin conjugate nanoparticles that abolish tumours after a single injection. Nat Mater 2009; 8: 9939.
  • 27
    Etrych T, Jelínková M, Ríhová B, Ulbrich K. New HPMA copolymers containing doxorubicin bound via pH-sensitive linkage: synthesis and preliminary in vitro and in vivo biological properties. J Control Release 2001; 73: 89102.
  • 28
    Ulbrich K, Etrych T, Chytil P, Jelínková M, Ríhová B. HPMA copolymers with pH-controlled release of doxorubicin: in vitro cytotoxicity and in vivo antitumor activity. J Control Release 2003; 87: 3347.
  • 29
    Storm G, ten Kate MT, Working PK, Bakker-Woudenberg IA. Doxorubicin entrapped in sterically stabilized liposomes: effects on bacterial blood clearance capacity of the mononuclear phagocyte system. Clin Cancer Res 1998; 3: 1115.
  • 30
    Drummond DC, Meyer O, Hong K, Kirpotin DB, Papahadjopoulos D. Optimizing liposomes for delivery of chemotherapeutic agents to solid tumors. Pharmacol Rev 1999; 51: 691743.