Results of multicenter double-blind placebo-controlled phase II clinical trial of Panagen preparation to evaluate its leukostimulatory activity and formation of the adaptive immune response in patients with stage II-IV breast cancer

Anastasia S Proskurina, Tatiana S Gvozdeva, Ekaterina A Alyamkina, Evgenia V Dolgova, Konstantin E Orishchenko, Valeriy P Nikolin, Nelly A Popova, Sergey V Sidorov, Elena R Chernykh, Alexandr A Ostanin, Olga Y Leplina, Victoria V Dvornichenko, Dmitriy M Ponomarenko, Galina S Soldatova, Nikolay A Varaksin, Tatiana G Ryabicheva, Peter N Uchakin, Stanislav N Zagrebelniy, Vladimir A Rogachev, Sergey S Bogachev, Mikhail A Shurdov, Anastasia S Proskurina, Tatiana S Gvozdeva, Ekaterina A Alyamkina, Evgenia V Dolgova, Konstantin E Orishchenko, Valeriy P Nikolin, Nelly A Popova, Sergey V Sidorov, Elena R Chernykh, Alexandr A Ostanin, Olga Y Leplina, Victoria V Dvornichenko, Dmitriy M Ponomarenko, Galina S Soldatova, Nikolay A Varaksin, Tatiana G Ryabicheva, Peter N Uchakin, Stanislav N Zagrebelniy, Vladimir A Rogachev, Sergey S Bogachev, Mikhail A Shurdov

Abstract

Background: We performed a multicenter, double-blind, placebo-controlled, phase II clinical trial of human dsDNA-based preparation Panagen in a tablet form. In total, 80 female patients with stage II-IV breast cancer were recruited.

Methods: Patients received three consecutive FAC (5-fluorouracil, doxorubicin and cyclophosphamide) or AC (doxorubicin and cyclophosphamide) adjuvant chemotherapies (3 weeks per course) and 6 tablets of 5 mg Panagen or placebo daily (one tablet every 2-3 hours, 30 mg/day) for 18 days during each chemotherapy course. Statistical analysis was performed using Statistica 6.0 software, and non-parametric analyses, namely Wilcoxon-Mann-Whitney and paired Wilcoxon tests. To describe the results, the following parameters were used: number of observations (n), median, interquartile range, and minimum-maximum range.

Results: Panagen displayed pronounced leukostimulatory and leukoprotective effects when combined with chemotherapy. In an ancillary protocol, anticancer effects of a tablet form of Panagen were analyzed. We show that Panagen helps maintain the pre-therapeutic activity level of innate antitumor immunity and induces formation of a peripheral pool of cytotoxic CD8+ perforin + T-cells. Our 3-year follow-up analysis demonstrates that 24% of patients who received Panagen relapsed or died after the therapy, as compared to 45% in the placebo cohort.

Conclusions: The data collected in this trial set Panagen as a multi-faceted "all-in-one" medicine that is capable of simultaneously sustaining hematopoiesis, sparing the innate immune cells from adverse effects of three consecutive rounds of chemotherapy and boosting individual adaptive immunity. Its unique feature is that it is delivered via gastrointestinal tract and acts through the lymphoid system of intestinal mucosa. Taken together, maintenance of the initial levels of innate immunity, development of adaptive cytotoxic immune response and significantly reduced incidence of relapses 3 years after the therapy argue for the anticancer activity of Panagen.

Trial registration: ClinicalTrials.gov NCT02115984 from 04/07/2014.

Figures

Figure 1
Figure 1
DNA concentration in blood plasma of healthy volunteers not receiving Panagen (control, n = 15) and following daily oral administration of 30 mg Panagen for 1 and 3 months (n = 9).
Figure 2
Figure 2
Dynamic changes in blood cell counts (×109cells/L) measured in the clinical trial at the initial pre-therapy timepoint (0) and on day 21 after each chemotherapy course. Median values in each group are shown. The number of patients per group is indicated for each time point. Significantly higher values are observed for Panagen (dashed orange line) vs placebo (black solid line) groups of patients (Wilcoxon-Mann–Whitney test), as well as within each group relatively to the initial level before the therapy (Wilcoxon paired test). For patients who received Panagen, increased value is marked with up-facing arrow, for patients from placebo group, decreased value is highlighted by downward-facing arrow. Red asterisk (*) denotes significant values with р < 0.05, blue hash symbol (#) marks statistically significant difference with р < 0.11.
Figure 3
Figure 3
Changes in stimulation indices (%) for blood cell types throughout three chemotherapy courses. Median values per group are shown. The number of patients per group is indicated for each time point. Stimulation index is expressed as a ratio of values measured in second and third control time points (days 14 and 21) to the appropriate value observed in the control point of the first chemotherapy course (set as 100%). Patients were subgrouped into Panagen-responders, Placebo and Panagen-non-responders. Red line denotes 100% level, i.e. the values reported in control time points (days 14 and 21) after the first chemotherapy. Values that show statistically significant difference between Panagen-responders and Placebo patient groups (Wilcoxon-Mann–Whitney test) with р < 0.01 (**), р < 0.05 (*) and р < 0.09 (#) are highlighted.
Figure 4
Figure 4
Frequency of grade I-IV neutropenia-related events in patients at base II on day 14 of three courses of FAC and AC chemotherapies.
Figure 5
Figure 5
Arbitrary content (%) of CD8+ perforin + cytotoxic T-cells in peripheral blood of patients at base II undergoing FAC or AC chemotherapy on day 21 after the first course, relatively to the initial baseline level (100%, red line). Panagen group is split to demonstrate that two distinct patient subgroups are present – “responders” (those having cell counts significantly different from the placebo group) and “non-responders”. Median values, quartile range 25-75% (box) and minimum-maximum range are given for each group; n – the number of patients per group. Significant differences from the Placebo group with p < 0.05 (Wilcoxon-Mann–Whitney) are marked with red asterisk.
Figure 6
Figure 6
Comparative analysis of cytotoxicity indices in PBMCs of patients (FAC regimen, base II) on day 21 following the third round of chemotherapy. Largely responsible for innate anticancer immunity, PBMCs retain their specific functions at the levels observed before the therapy in patients receiving Panagen throughout three courses of chemotherapy (р < 0.05). Unlike in Panagen cohort, PBMCs from placebo group patients display three-fold decrease in cytotoxicity indices by the end of the third chemotherapy course relatively to the initial level.
Figure 7
Figure 7
Effects of Panagen on patient cytokine profiles. Comparison of spontaneous and mitogen-stimulated cytokine secretion in Control (n = 4) and Panagen-treated (n = 12) patients under FAC chemotherapy regimen and in Control (n = 6) and Panagen-treated (n = 19) patients under AC chemotherapy regimen. T0 – before the therapy, Т1 – day 21 after the first chemotherapy, Т2 – day 21 after the third chemotherapy. Results were analyzed by One Way Repeated Measures and Two Way ANOVA with post hoc Holm-Sidak and Tukey tests in order to evaluate significance of interval-dependent changes, as well as difference between groups. Absolute units of measure (pg/ml) were converted to natural logarithms in order to normalize data prior to analysis. Data presented as Mean ± SEM; a,b – statistically significant difference (p < 0.05) vs T0 or T1 interval respectively; c – statistically significant difference (p < 0.05) between Panagen and Control groups.
Figure 8
Figure 8
3-year follow-up analysis of Panagen clinical trial. Percentage of relapse events and deaths of patients relatively to the total number of patients. Data for patients from the base II (FAC and AC regimen) are presented. Each group of bars is labeled to show the percentage of patients having stage II, III or IV cancers.

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Source: PubMed

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