3 Results and Discussion
3.3 HPMA Copolymers: Therapy Study
Based on the promising tumor accumulation studies with HPMA copolymers, a therapy study was planned and carried out with the star-like HPMA copolymer (200 kDa) containing pH-dependent releasable doxorubicin. DLD-1 was chosen as tumor model because of its chemotherapy resistance to doxorubicin and because of the higher accumulation of HPMA copolymers in this model in the previous experiments.
Chemotherapy resistance means that an inhibitory effect on the tumor cannot be achieved in vitro with a concentration of the cytostatic agent that corresponds to human peak plasma levels. Treatment with DOX-HPMA conjugates has been already shown to be effective in EL4-T-cell lymphoma previously, but not yet in doxorubicin-resistant colon carcinomas.239,240 To keep the number of animals manageable, this study was focused on the star-like architecture. A conjugate of doxorubicin with the star-like HPMA copolymer (200 kDa) was synthetized by the cooperation partners in Prague. In vitro release studies demonstrated comparable stability of doxorubicin-DY-676 and DY-676-PYR in pH 7.4 buffer and release in pH 5 buffer.238 This suggests a similar in vivo behavior of the therapy polymer and the dual fluorescent star-like HPMA containing the S-PYR spacer.
Within this therapy study, five groups were compared (cp. Table 3, p. 25): One group was conventionally treated with doxorubicin at a concentration of 5 mg/kg body weight, as the maximum tolerated dose (MTD) reported for doxorubicin in mice ranges from 5-10 mg/kg body weight.241-244 Two groups were treated with HPMA-DOX conjugates, one with an equivalent dose and the other with a 3-fold increased dose. The remaining two groups served as control groups. A weekly dosing regimen was planned, but the treatment was halted for groups showing severe toxicities (e.g. weight loss, atypical behaviour, pronounced skin reactions).The results of the therapy study are presented in Figure 31. Both control groups showed exponential tumor growth and no weight loss.
With respect to the rapidly increasing tumor size and tumor burden these mice were sacrificed 36 days after tumor inoculation.
h Polymer synthesis: Institute of Macromolecular Chemistry AS CR, Prague, Czech Republic (T. Etrych, and P. Chytil). Study design: Stefan Hoffmann in cooperation with the Department of Internal Medicine IV, Oncology/Hematology, Martin-Luther-University Halle-Wittenberg (H. Caysa and T. Mueller). Study realization: Stefan Hoffmann, T. Mueller and A.-K. Heinrich (Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg). Data Evaluation: Stefan Hoffmann.
3 Results and Discussion HPMA Copolymers: Therapy Study 68
Figure 31. A: Injections during the therapy study. Tumors were inoculated at day 0.
Therapy was administered once weekly taking toxicity into consideration. Only groups showing no severe toxicity or recovery from toxicity were re-treated in the next cycle. B:
Tumor growth during treatment. Exponential growth can be observed for all groups but the DOX-HPMA group, which showed a stable mean tumor volume during the intensified therapy regimen (twice weekly) between day 40 and 55. C: Mouse weight was measured daily during therapy as one indicator for doxorubicin toxicity. The weight of the control groups did not decrease and is therefore omitted here. Interestingly the weight of the DOX-HPMA group did not decrease during weekly therapy, indicating low toxicity. All data represent means of six animals ± SD.
3 Results and Discussion HPMA Copolymers: Therapy Study 69 The “3x DOX-HPMA”-group and the “Free-DOX”-group showed marked toxicity, which led to cessation of the weekly planned dosing regimen after only one or two injections, respectively. The pronounced toxicity in the group treated with free doxorubicin is remarkable, as higher MTDs were reported in literature.241-244 Toxicity included atypical behavior, weight loss and doxorubicin-typical skin reactions and led to cessation of the experiment for the “3x DOX-HPMA”-group 13 days after one single injection only. The mice in the group treated with free DOX recovered within two weeks after the second injection, but due to cessation of the therapy an increasing tumor size was observed in this group, which led to the end of the experiment at day 44 without any further injection.
Contrary to the “free DOX”-group, neither weight loss nor other signs of toxicity were observed in the group that was treated with the DOX-HPMA (equivalent doxorubicin dose) during the weekly dosing regimen. This effect can be explained by the lower amount of free doxorubicin circulating in the blood stream and the more local release of the active DOX in the tumor.
The overall good therapy tolerance in the DOX-HPMA group led to a change of the dosing regimen to twice weekly (accordingly 10 mg/kg per week), which subsequently resulted in weight loss of the mice, but also in a stable tumor size (mean) in the doxorubicin-resistant colon carcinoma xenograft tumor model (Figure 31). However the individual therapy response was varying (Table 7).
Table 7. Change of the tumor volume in cm3 of the mice treated with DOX-HPMA during 14 days of the weekly dosing regimen (from day 26 – day 40) and during the twice weekly dosing regimen (from day 40 – day 54).
Mouse Day 26 Day 40 Day 54
1 0.63 0.98 (+56 %) 1.15 (+17 %)
2 0.79 1.01 (+28 %) 1.05 (+4 %)
3 0.55 0.65 (+18 %) 0.51 (-22 %)
4 0.35 0.40 (+14 %) 0.53 (+33 %)
5 0.76 0.95 (+25 %) 0.76 (-20 %)
6 1.13 1.64 (+45 %) 2.00 (+22 %)
The tumors in the DOX-HPMA group grew slower than in the control group, which was treated conventionally with doxorubicin. The tumor volume distribution in both groups was not comparable, likely because the sample size of 6 animals per group is small.
Thus the non-parametric Mood’s median test was used to demonstrate a statistically
3 Results and Discussion HPMA Copolymers: Therapy Study 70 significant difference between the DOX-HPMA group and the “free DOX” group 42 days after tumor cell inoculation (p=0.02) Although the tumor volume distributions are not comparable between both groups and the interindividual variation is high, the Mood’s median test and the tumor volume trend during the experiment (presented in Figure 32) suggests a superior efficacy of the DOX-HPMA. However, this result needs further confirmation with an increased number of animals, which would allow more powerful statistical testing.
Figure 32. Tumor volume distribution within the DOX-HPMA group and the “free DOX”
group 28 days (A) or 42 days (B) after tumor inoculation, respectively. A superior effect of the DOX-HPMA can be expected from the tumor volume trend. Boxes represent median, 1st and 3rd quartile; whiskers represent minimum and maximum; squares represent the mean.
The results of the therapy study demonstrate an additional therapeutic effect of the DOX-HPMA copolymers. Compared to the same dose of free DOX, a reduced toxicity was observed and thus a higher dose could be administered. Considering the aggressive and fast growing doxorubicin-resistant tumor model, the results are likely to be even more impressive when sensitive tumor models or tumors with slower growth rate are used, which should be addressed in further experiments.
3 Results and Discussion Carbohydrate Plasma Volume Expanders 71