The first step was to assess the acute toxicity of the medical product "Activegel" in vivo to understand and confirm the data on the test substance (stated by the manufacturer) on the absence of its toxicity. During the experiment, the survival and physiological parameters of rats (males and females) in the control and experimental groups were analyzed. During 48 hours (stage I) and 14 days (stage II) we have analyzed locomotor and respiratory activity of experimental animals, the condition of fur, mucous membranes, behavior, usage of food and water, and gastrointestinal function.
We have found that the highest of the studied doses of “Activegel” (5000 mg/kg) did not cause the death of animals. In the control and all experimental groups of male rats within 48 and 14 days after the 1st injection of the test substance, no deviations in the studied indices of the physiological state were observed. However, immediately after injection of saline or test substance and for the next 15 minutes in 50-100% of the animals the signs of aggression were observed. Since aggressive behavior of animals was observed in both control and experimental groups, it is clear that it was not a reaction to the test substance, but to the procedure of forced fixation of rats for injection procedure. In groups of female rats, which were administered different doses of "Activegel" or saline, no violations of the physiological state of the experimental animals were observed during the entire observation period.
Analysis of the dynamics of body weight changes in rats within 48 hours and 14 days after single subcutaneous injection of "Activegel" at different doses did not reveal statistically significant differences in the studied index compared with the control group: animal body weight in the experimental groups increased as well as in the control.
Macroscopic examination of the condition of the internal organs of experimental animals and the condition of the tissues at the injection site after single subcutaneous administration of "Activegel" at different doses showed that it did not affect the internal organs of the animals.
One of the most important stages of the study of medicinal products is the macroscopic analysis of the condition of the tissues located in the injection site of the test substance, because the characteristics of such contact areas are indicative of its aggressiveness. We showed the absence of signs of inflammation and abnormalities in the condition of the studied tissues in experimental animals (Fig. 1, 2).
Pathomorphological examination of skin samples of all experimental animals of both sexes showed no pathological changes: no redness, hemorrhage, rupture, pus, edema, the presence of foreign bodies etc (Fig. 3 - 6). Morphological examination showed normal histoarchitectonics of the skin in all groups of animals of both sexes. In the skin there was a clear division into the epidermis and the dermis. Under the skin, subcutaneous fat and a layer of the skin muscle fibers were visible. The epidermis was represented by a multilayered keratinized epithelium lying on a distinct basement membrane that was uniform in thickness, without ruptures or thinnings. Maturity and zonation of the basal, prickle and granular layers of the epidermis without signs of inflammatory infiltrate were clearly traced. The papillae of the papillary layer of the dermis of all experimental groups were clearly expressed and deeply immersed in the epithelial layer. In the papillary layer there was loose connective tissue with vessels without pathological signs. No signs of inflammation in the papillary layer were found in any of the experimental groups.
The reticular layer of the dermis is represented by a dense unformed connective tissue. Fibroblasts were present in significant quantities in the dermis of all test specimens; the number of fibroblasts and collagen fibers indicates active collagenosis. There were no signs of edema in the reticular layer. By the nature of collagen fibers, no signs of scarring or fibrosis were detected, and the blood supply to the skin of all experimental animals was normal. The condition of the vessels of the dermis was normal, without hemostasis, signs of dystonia or spasms: no vascular hyperemia, vascular endothelium is not changed, not thickened. No inflammatory lymphocyte infiltration (neither diffuse nor local infiltration) was detected in the papillary and reticular layers of the dermis in any sample at all observation time points. In addition, the absence of eosinophils and macrophages in the dermis indicated the absence of an allergic reaction to the implant.
In the reticular layer of the dermis of all experimental animals a large number of mature hair follicles were preserved. The follicles were not deformed, without signs of keratinocyte hyperplasia. Next to the follicles there were often visible blood vessels, the endothelium of which exerted no pathological changes. Also, all groups of animals had a large number of sebaceous glands, the structure of which corresponded to the physiological norm. The hypodermis was formed by clusters of adipocytes without signs of edema or redness.
The obtained results indicated a normal morpho-functional state of the skin tissues of experimental animals of both sexes at the location of the implanted substance irrespectively of the doses and timing of implantation.
Thus, according to the study of in vivo toxicity of "Activegel", no clinical manifestations of intoxication were observed in animals of all experimental groups: no deviations in the studied indices of the physiological state of rats, changes in the weight of animals and their internal organs, or disorders of their internal organs. The normal condition of animal skin tissues at the injection site and the absence of systemic and local markers of inflammation were registered.
The results of general and biochemical blood tests of experimental animals of both sexes 48 hours after injection of the test substance indicated the absence of significant changes in most of the studied indices. However, in the group of male rats injected with “Activegel” at a dose of 2000 mg/kg, biochemical analysis of peripheral blood revealed a significantly lower glucose concentration compared to the control. In the group of male rats treated with "Actigel" at a dose of 500 mg/kg, there was registered a significantly lower ALT activity (by 16.2%) compared with the control (р < 0.05). It is important to note that the above changes in indices, although different from the control group, were within normal range [19-22].
Assessment of the effect of "Activegel" on the indices of general and biochemical blood tests of female rats revealed a significant increase in the leukocyte counts (by 26.4%, compared with the control, p ˂ 0.05) only in the group treated with the highest dose (5000 mg/kg). It should be noted that in this group of animals the increase in the leukocyte count was not accompanied by changes in the leukocyte formula. Therefore, the results suggest that this is a physiological leukocytosis, a temporary phenomenon not associated with disorders of the body.
In all groups of female rats administered with “Activegel”, an increase in the concentration of inorganic phosphorus was observed by 31% (5000 mg/kg) (р < 0.05), 25% (2000 mg/kg) (р < 0.05) and 37.5% (500 mg/kg) (р < 0.05) compared with the control group.
In the group of female rats injected with the test substance at a dose of 500 mg/kg, a statistically significant decrease in the concentration of urea in the blood by 15% (р < 0.05) was registered, compared with the control group.
The results of general and biochemical blood tests of animals of both sexes 14 days after single injection of the test substance indicated the absence of significant changes in most of the studied indices. However, in the group of male rats injected with the test substance at a dose of 5000 mg/kg, a significant (р ˂ 0.05) increase in glucose concentration by 26.3% and ionized calcium by 9% was observed, compared with the control group. In the group of male rats injected with "Activegel" at a dose of 2000 mg/kg, general and biochemical analysis of peripheral blood revealed the significantly higher concentrations of hemoglobin (by 6.8%), glucose (by 26.3%) and ionized calcium (by 9%) compared to the control. In the group of male rats administered with "Activegel" at a dose of 500 mg/kg we registered a significantly lower hemoglobin concentration (4.8%) and higher glucose concentration (31.6%) compared with the control. One should note that the above changes in the indices of general and biochemical blood analysis of of rats, although different from the control group, remained within normal range [19, 21, 23, 24].
It should be noted that in the groups of male rats administered with the test substance at doses of 2000 mg/kg and 500 mg/kg, there was also observed a significant increase in the concentration of total bilirubin by 35.7% (р < 0.05) and 42.9% (р < 0.05), respectively, compared with the control. As far as in animals of the these groups moderate but significant differences in hemoglobin concentration were registered, it’s reasonable to suppose that the increase in total bilirubin concentration could be associated with physiological processes of hemoglobin synthesis/breakdown in experimental animals.
According to the results of the general blood analysis of female rats, a significant increase in the leukocyte counts by 34.5% and two-fold increase of monocyte counts was found compared with the control, in animals treated only with the largest of the studied doses (5000 mg/kg) of the test substance. In this group of animals, leukocytosis was accompanied by changes in the leukocyte formula, in particular, a small increase in the % content of monocytes, which may indicate the reactivity of the organism in response to the introduction of a large dose of test substance.
In the group of female rats injected with Activegel at a dose of 2000 mg/kg, biochemical analysis of peripheral blood revealed only a significantly higher concentration of ionized calcium by 9%, compared with the control group. In the group of female rats injected with the test substance at a dose of 500 mg/kg a significantly lower glucose concentration and higher concentration of inorganic phosphorus (46.7%) was observed compared with the control. It is important to note that these altered biochemical parameters, according to the literature, were within normal range [21, 24].
Thus, the in vivo toxicity of the new generation hydrophilic gel was evaluated using generally accepted approaches to clinical trials of the compounds and it was shown that this substance, according to the classification of the Environmental Protection Agency can be classified as hazard category IV, is not toxic. The data obtained by us can be used for further preclinical and clinical studies on the medical use of new generation hydrophilic gel "Activegel".
As the lack of toxicity of the test substance in vivo was shown, to expand the understanding of its safety, certain in vitro studies of “Activegel” have been planned. The next step was to study its possible genotoxic effect in vitro using the analysis of human peripheral blood lymphocytes by DNA comet assay after their exposure to the test substance.
Analysis of the DNA integrity in peripheral blood lymphocytes of healthy donors cultured in the presence of “Activegel” at a concentration of 0.2 g of the substance per 1 ml of nutrient medium showed that the test substance exerted no genotoxic effects on the cells. The studied parameters of DNA comets in the experimental group (the amount of DNA in the "tail" of comets, the length and moment of the tail) did not differ from the parameters of the control (cells treated with saline) (Table 1, Fig. 7, 8).
Table 1
Analysis of the parameters of DNA comets of human peripheral blood lymphocytes after their cultivation in the presence of a medical product "Activegel" in vitro
Experimental group
|
Parameters of DNA comets
|
Tail length, px
|
% DNA in tail
|
Tail moment
|
Peripheral blood lymphocytes + saline, Control
|
Donor №1
|
85.71 ± 5.6
|
0.25 ± 0.02
|
0.27 ± 0.03
|
Donor №2
|
97.31 ±2.0
|
0.20 ± 0.01
|
0.25 ± 0.02
|
Donor №3
|
82.07 ± 3.5
|
0.17 ± 0.01
|
0.20 ± 0.02
|
Donor №4
|
83.52 ± 6.6
|
0.20 ± 0.05
|
0.21 ± 0.01
|
М ± m
|
87.2 ± 6.9
|
0.21 ± 0.03
|
0.23 ± 0.03
|
Peripheral blood lymphocytes + “Activegel”
|
Donor №1
|
93.96 ± 3.0
|
0.22 ± 0.02
|
0.22 ± 0.01
|
Donor №2
|
72.96 ± 8.5
|
0.15 ± 0.02
|
0.23 ± 0.0
|
Donor №3
|
96.97 ± 0.4
|
0.19 ± 0.02
|
0.22 ± 0.09
|
Donor №4
|
104.14 ± 17.1
|
0.27 ± 0.12
|
0.21 ± 0.04
|
М ± m
|
92.0 ± 13.4
|
0.21 ± 0.05
|
0.22 ± 0.01
|
In the experiment, a small "tail" of the comet was observed, which evidenced on of the presence of a low number of single-stranded DNA breaks. However, the low level of luminescence and the large area of the "tail" of the comet are characteristic of cases where nucleic acid breaks can be attributed to "junk DNA", which is a variant of the norm. Therefore, these results indicated that "Activegel" in the studied concentration (0.2 g/ml) did not affect the parameters of DNA comets, ie the test substance exerted no genotoxic effect on the cells.
The obtained data on the absence of genotoxicity on human lymphocytes are an additional positive characteristic of the new generation hydrophilic gel for further expanding of its use. Because this substance can potentially be used as an implant in breast reconstruction in breast cancer patients, we conducted the study on the effect of “Activegel” on breast cancer cells of varying degrees of malignancy, MCF-7 and MDA-MB-231 cell lines. Moreover, this hydrophilic gel (as well as its known analogues) can be potentially used as a "vector" for targeted delivery of biologically active molecules, including anticancer drugs, therefore an assessment of its effect on tumor cells is really important. In this work, we have studied the effect of breast cancer cells exposure to the gel on their proliferative activity (Table 2, 3).
Table 2
The number of living cells of MDA-MB-231 line after the action of "Activegel"
% of conditioned nutrient medium
|
MDA-MB-231 + saline solution
|
MDA-MB-231 + “Activegel”
|
№1
|
№2
|
№3
|
№1
|
№2
|
№3
|
Number of living cells, %
|
|
48 h
|
100
|
100.4±1.8
|
102.7±3.2
|
103.6±3.5
|
104.3±3.9
|
102.2±4.3
|
99.7±3.8
|
50
|
104.0±4.4
|
106.1±0.2
|
98.0±0.9
|
100.4±3.7
|
101.8±4.7
|
101.9±2.4
|
25
|
101.6±3.5
|
103.3±3.1
|
102.0±3.1
|
103.1±2.9
|
103.3±2.8
|
100.5±2.1
|
12.5
|
100.7±0.9
|
100.8±5.0
|
99.6±4.4
|
101.5±3.8
|
103.9±4.1
|
100.8±3.1
|
6.3
|
101.9±1.0
|
102.5±3.5
|
101.0±2.8
|
101.0±3.8
|
103.4±1.9
|
99.6±1.8
|
3.1
|
103.2±2.6
|
102.1±2.8
|
101.7±3.4
|
100.6±2.3
|
103.2±1.8
|
102.8±2.6
|
1.6
|
104.4±2.2
|
103.3±2.3
|
99.3±1.8
|
99.6±0.7
|
100.9±3.1
|
100.5±1.6
|
|
96 h
|
100
|
100.8±2.3
|
100.0±3.0
|
101.7±3.7
|
98.3±1.1
|
100.4±3.6
|
99.1±3.2
|
50
|
99.0±4.1
|
99.7±1.6
|
100.1±5.0
|
100.9±4.1
|
102.1±3.1
|
101.4±2.7
|
25
|
100.9±3.6
|
98.7±3.0
|
104.6±1.5
|
102.7±2.5
|
103.1±2.0
|
101.1±5.0
|
12.5
|
101.7±3.2
|
100.6±0.9
|
103.7±2.6
|
102.5±3.0
|
103.1±1.5
|
102.7±2.4
|
6.3
|
99.5±1.8
|
100.9±1.9
|
101.5±3.6
|
102.7±3.8
|
101.4±2.9
|
101.0±2.2
|
3.1
|
102.8±2.9
|
100.6±4.0
|
102.5±1.9
|
102.4±3.6
|
99.0±2.9
|
102.3±2.6
|
1.6
|
98.3±2.5
|
102.9±2.2
|
99.9±2.6
|
101.1±2.1
|
99.6±1.2
|
98.7±3.1
|
Table 3
The number of living cells of MCF-7 line after the action of "Activegel"
% of conditioned nutrient medium
|
MCF-7 + saline solution
|
MCF-7 + “Activegel”
|
№1
|
№2
|
№3
|
№1
|
№2
|
№3
|
Number of living cells, %
|
|
48 h
|
100
|
101.3±2.3
|
97.2±1.5
|
100.3±0.5
|
101.5±2.4
|
101.1±3.0
|
99.0±1.7
|
50
|
99.9±0.8
|
99.9±2.3
|
98.7±1.6
|
100.0±0.9
|
100.6±2.7
|
105.0±1.8
|
25
|
99.9±2.9
|
100.3±3.2
|
102.3±2.7
|
98.1±1.1
|
101.2±1.4
|
100.2±0.7
|
12.5
|
100.8±1.9
|
99.6±0.5
|
99.2±1.8
|
100.3±3.2
|
99.3±3.6
|
101.0±1.8
|
6.3
|
101.7±3.2
|
101.3±2.9
|
100.0±1.9
|
99.7±2.0
|
101.8±4.2
|
102.6±2.2
|
3.1
|
99.3±2.5
|
102.2±3.1
|
99.9±1.5
|
101.6±1.4
|
99.2±1.5
|
101.4±2.7
|
1.6
|
99.3±2.8
|
101.6±1.1
|
98.7±1.4
|
100.1±2.2
|
101.9±2.3
|
98.7±3.7
|
|
96 h
|
100
|
100.3±2.0
|
99.6±2.1
|
100.3±1.8
|
99.8±3.1
|
99.1±1.0
|
99.4±3.1
|
50
|
102.2±2.1
|
99.0±3.4
|
99.7±0.5
|
101.0±3.1
|
99.2±1.7
|
100.4±2.7
|
25
|
99.5±2.1
|
101.0±1.3
|
103.5±1.3
|
100.0±1.0
|
101.3±1.3
|
100.1±0.4
|
12.5
|
101.0±2.2
|
102.0±2.1
|
100.8±2.6
|
102.8±1.4
|
98.4±1.2
|
101.9±2.8
|
6.3
|
100.5±2.0
|
102.0±2.2
|
101.2±1.9
|
99.4±2.9
|
102.3±3.3
|
101.7±2.9
|
3.1
|
101.3±2.9
|
103.2±1.6
|
100.9±2.8
|
99.7±1.5
|
100.0±0.2
|
103.2±2.3
|
1.6
|
98.3±3.7
|
104.0±3.0
|
103.4±2.2
|
98.0±2.2
|
99.2±1.2
|
99.0±2.5
|
According to the study, it was determined that the new generation hydrophilic gel for implantation does not affect either the proliferative activity (Table 2) or the viability of cells (Table 3) of human breast cancer in the in vitro system, which potentially indicates its inertness ( but requires further more detailed clinical trials when used as an implant after surgery for patients with a history of breast cancer).
There was no difference between the effect of the test gel on malignantly transformed cells of varying degrees of malignancy: the inertness of the action of "Activegel" was observed when assessing the growth characteristics of both cell lines (MCF7, and MDA-MB-231). The findings are highly important because they indicate the potential safety of this product in cancer patients, but such statements require further more detailed study of the effect of "Activegel" on tumor cells (including their phenotypic characteristics and tumorigenicity). Such research is important, because the revealed lack of toxicity of the hydrophilic gel and the inertness of its action on tumor cells opens up new possibilities for the use of this product as a vector for targeted therapy.
In our opinion, this direction of application of hydrophilic gel ("Activegel") looks quite promising, as the known ability of hydrogel to encapsulate and release small molecules and biologicals is currently being actively implemented for synthetic preparations (used as a "depot system with targeted delivery"). In particular, there are known such developments as “Endo's Vantas ®” approved by the FDA for subcutaneous hormone therapy to prevent the growth of hormone-dependent prostate cancer cells, and “SpaceOAR®” hydrogel (Augmenix) for the protection of prostate cancer patients undergoing radiation therapy [14].
However, to date, none of the natural hydrogels (as opposed to synthetic) have been approved as targeted delivery systems for antitumor drugs, which may be due to the advantages of synthetic hydrogels due to the possibility of prolonged release of bioactive molecules, i.e. significant prolongation of local action. The benefits of using synthetic hydrogels are also evidenced by the data from “TraceIT®” and “SpaceOAR®”, which are widely studied to visualize tumor cells and protect normal cells from radiation damage [14].