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THE EFFECTIVENESS OF SODIUM DEOXYRIBONUCLEATE IN THE COMPLEX TREATMENT OF VICTIMS WITH SEVERE ASSOCIATED INJURY: A PROSPECTIVE, RANDOMIZED, PLACEBO-CONTROLLED STUDY Tulupov A.N., Dulaev A.K., Gromov M.I., Pivovarova L.P., Lapshin V.N., Sinenchenko G.I., Nikitin A.V., Fedorov A.V., Markelova E.V., Osipova I.V., Razumova N.K.

Saint Petersburg I.I. Dzhanelidze Research Institute of Emergency Medicine,

Saint Petersburg, Russia

 

A common feature of the course of severe associated injury (AI) is insufficiency of immune and hematopoietic system, and cellular metabolism disorder which develop in the period of acute response to trauma [1]. The clinical manifestation of such disorders is development of complications of infectious and non-infectious origin. The incidence of infectious complications after polytrauma reaches 90 % for pneumonia, and 26-52 % for sepsis [2]. The laboratory findings testify the development of acute posttraumatic anemia, hypoproteinemia and secondary immune insufficiency [3].

The standard treatment of acute posthemorrhagic anemia and hypoproteinemia supposes the replacement therapy with donor blood components. However, such treatment causes the pulmonary vascular microembolization and immune suppression, is limited by donorship resources and is hazard due to risk of various viral infections [2].

Various immune modulators were used for correction of secondary immune deficiency in the associated injury [4]. Recombinant interleukin-2 [5], and the combination of interleukin-2 and sodium deoxyribonucleate [6] were efficient for patients with sepsis and lymphocytopenia.

The ability of various nucleic acids for activation of tissue healing is known from 1960s [7]. A recent experimental study showed that the use of native fragments of sodium deoxyribonucleate as local application decreased the edema and injury to the skin in region of bedsores by means of suppression of local oxidative stress caused by tissue ischemia-reperfusion [8].

Objective – to estimate the effect of deoxyribonucleic acid on the incidence of complications and the duration of treatment, as well as the state of immunity and hematopoiesis in victims with severe associated injury.

 

MATERIALS AND METHODS

Derinat© (Tekhnomedservis, Russia, registration number No. R N002916/01) presents the low molecular native fragments of DNA in view of sodium deoxyribonucleate from milt of sturgeons. It realizes the reparative and immunomodulating action by delivery of bioavailable polynucleotides to the body. The high accumulation and consumption of the agent happens in the most actively dividing cells – in bone marrow, the spleen, lymphatic nodes, the skin and mucosa [7].

The inclusion criteria to the study were the patients’ age of 18-70, traumatic shock of degree 2 or 3, with probable mortality < 50 % and the value of predictive criterion ±Т within the range from +7.5 to +48 hours [9]. The exclusion criteria were concurrent sexually transmitted diseases, viral hepatitis, HIV, chronic renal insufficiency, chronic diseases requiring for anti-inflammatory, hormonal and cytostatic drugs, chronic alcoholism, drug addiction, pregnancy. The exclusion from the study group was late identification of the exclusion criteria, and transfer of patients to other hospitals.

The study design corresponded to conditions of prospective, double blind, placebo controlled study. The patients and medical staff did not know where bottles with the active drugs were or where placebo was. Each patient received the contents of a flacon (5 ml) designated as Derinat (75 mg) of the same series. The intramuscular injection was made one time a day during 10 days beginning from next day post injury. The patients were randomized into two groups according to generation of 60 random numbers. The even number corresponded to the even series of the drug, the non-even number was the non-even series of production. The placebo was of identical appearance and other numbers of production series.

The study included 60 patients (47 men and 13 women at the age of 20-68) with severe closed associated mechanic injuries who were admitted immediately from Saint Petersburg I.I. Dzhanelidze Research Institute of Emergency Medicine from May, 2016, to May, 2018. 83 % of patients were persons of working age. The severity of injuries was estimated with ISS [10], the severity and the probable outcome of traumatic shock (+T) – with the formula [9], the severity of organ dysfunction – with SOFA [11]. Sepsis categories were determined in concordance with the recommendations of Chicago Consensus Conference [12].

All patients received the clinical, laboratory, radiologic and instrumental examinations, as well as the treatment in compliance with the present order for arrangement of medical care for patients with associated, multiple and single injuries with shock. Additionally, each patient was estimated for blood levels of mononuclear cells expressing the receptor of stem cell factor (CD117), and neutrophilic granulocytes (NG), which contents the antimicrobial peptides of human defensin (def⁺NG).

The main directions for anti-shock treatment included the recovery of oxygen transport (infusion, transfusion, cardio- and vasoactive therapy, respiratory therapy), correction of consequences of hypoxia and reperfusion (antioxidant and antihypoxant therapy, proteolysis inhibitors), correction of hemostasis, antibacterial, nutritive and detoxication therapy [2].

The prognosis (with ±Т criterion) was unclear in all patients admitted to the anti-shock surgery room. As result, only critical care operations (primary interventions according to Damage control) and low-invasive medicodiagnostic procedures were carried out. After anti-shock treatment and stabilization of vital functions, the early restorative interventions were realized in full volume in the manner, which allowed performing the procedures with the first surgical “window of opportunities”, i.e. before formation of infectious complications, which demonstrate the highest incidence on the days 3-7 after trauma. For the whole period of the treatment in the trauma center, the patients of the main group received 4 various surgical interventions on average, the patients of the comparison group – 5.

The complications were considered as life-threatening and non-life-threatening, and infectious and non-infectious. The condition of erythroid lineage of hematosis (erythron) was monitored with the data of clinical blood analysis (Sysmex XT-000i). Protein-synthesis function of cells was monitored with the level of total protein in blood serum (Cobas c501). The number of days was calculated when the laboratory signs of anemia and hemoglobin < 100 and < 90 g/l, and hypoproteinemia with total protein < 60 and < 50 g/l were registered.

The study protocol was approved by the local ethical committee. The informed consent for participation in the study was received from each patient.

The group of healthy individuals (n = 15) of the same age and gender was used for comparison of erythropoiesis and total protein of the blood.

The statistical preparation of the data was realized with Statistica 6.0. The description statistics of the groups is presented by the average value and error of the mean, and by calculation of percentage proportions. Non-parametrical tests were used for estimation of quantitative values: Mann-Whitney’s test – for intergroup comparison, Wilcoxon’s test – for intragroup comparison. The analysis of nominal data was performed with Fisher’s exact test. The correlation was analyzed with Pearson’s parametrical method. P value < 0.05 was statistically significant.

The final cohort for analysis included 54 patients including 27 patients in the main group (with the studied agent), and 27 patients in the comparison group (placebo). 6 patients were excluded from the study (transfer to other hospital, pregnancy).

 

RESULTS AND DISCUSSION

According to the tables 1-3, the patients of both groups did not demonstrate any significant differences in gender, age, ISS, shock severity (±Т), characteristics of injuries, and techniques of surgical and conservative management.

Table 1

General characteristics of patients with associated injury

Values	Main group	Comparison group
Amount of patients	27	27
Men (%)	21 (78 %)	20 (74 %)
Age, years	39 (29; 51)	40 (26; 53)
Road accident (%)	21 (78 %)	19 (70 %)
Catatrauma  (%)	5 (19 %)	6 (22 %)
Industrial injury (%)	1 (4 %)	2 (7 %)
Shock of degree 2 (%)	17 (63 %)	16 (59 %)
Shock of degree 3 (%)	10 (37 %)	11 (41 %)
ISS, points	26.0 ± 1.2	24.7 ± 1.2
±T, hours	+12.9 ± 0.9	+12.0 ± 0.7
Total number of hemotransfusion, l	1.8 ± 0.3	1.8 ± 0.2
ICU stay, days	5.9 ± 0.9	6.2 ± 1.0
Number of days with SOFA > 0	4 3 ± 0.7	4.5 ± 0.7

 

Table 2

Incidence of injuries to various regions of the body

Region	Incidence of injuries
	General		Dominating injuries		Concurrent injuries
	Group		Group		Group
	Main	Comparison	Main	Comparison	Main	Comparison
Head	52 % (14)	67 % (18)	14.8 % (4)	7.4 % (2)	37 % (10)	14.8 % (4)
Chest	52 % (14)	52 % (14)	40.7 % (11)	44.4 % (12)	14.8 % (4)	22.2 % (6)
Abdomen	11 % (3)	26 % (7)	7.4 % (2)	11.1 % (3)	0	11.1 % (3)
Pelvis	33 % (9)	30 % (8)	18.5 % (5)	18.5 % (5)	7.4 % (2)	0
Spine	44 % (12)	37 % (10)	3.7 % (1)	0	14.8 % (4)	7.4 % (2)
Extremities	44 % (12)	44 % (12)	14.8 % (4)	14.8 % (4)	7.4 % (2)	7.4 % (2)
2 regions	15 % (4)	19 % (5)	0	0	14.8 % (4)	18.5 % (5)
3 and more regions	85 % (23)	81 % (22)	0	0	0	3.7 % (1)

 

Table 3

Incidence of injuries to organs and structures in polytrauma in groups

Injury pattern	Group
	Main	Comparison
Closed traumatic brain injury	52 %	67 %
Facial bone fractures	19 %	41 %
Lung contusion and laceration	81 %	67 %
Multiple rib fractures	41 %	59 %
Liver laceration	2 %	4 %
Spleen laceration	11 %	7 %
Laceration and contusion of small intestine and its mesentery	7 %	4 %
Laceration and contusion of colon and its mesentery	4 %	2 %
Kidney contusion and laceration	1 %	4 %
Urine bladder and urethra laceration	2 %	7 %
Pelvis fractures	33 %	30 %
Spine fractures	44 %	37 %
Humerus fractures	19 %	4 %
Forearm fractures	7 %	22 %
Hip fractures	30 %	22 %
Leg fractures	37 %	11 %

The estimation of severity of patients’ condition during treatment in ICU showed the absence of differences in mean duration of organ function disorders (SOFA > 0), and duration of stay in the unit. Also there were not any differences in average volume of hemotransfusion.

Acute posthemorrhagic anemia of mild degree [13] (normochromic, normocytic, regeneratory), and hypoproteinemia were registered in both groups on the following day after trauma (the table 4).

Table 4

Laboratory characteristics of erythropoiesis and protein level in blood of patients with associated injury (1 day after injury)

Values	Groups
	Healthy	Main group	Comparison group	Р1,2        P1,3       P2,3
	1	2	3
Red blood cells, 1012/l	4.6 ± 0.2	3.5 ± 0.1	3.3 ± 0.1	0.000   0.000   0.163
Hemoglobin, g/l	139 ± 7	107 ± 3	104 ± 4	0.002   0.001  0.551
Mean level of hemoglobin in erythrocyte, pg	29.6 ± 1.0	30.7 ± 0.2	30.8 ± 0.4	0.332   0.312  0.824
Reticulocytes, %	0.94 ± 0.22	1.24 ± 0.10	1.10 ± 0.10	0.223   0.512    0.327
Hematocrit, %	41.8 ± 2.1	31.6 ± 0.8	31.1 ± 1.1	0.001   0.001   0.715
Total protein, g/l	70.8 ± 3.2	56.3 ± 1.0	53.9 ± 1.5	0.000   0.000    0.189

Note: p – reliability of differences according to Mann-Whitney’s test.

Subsequent treatment of polytrauma was analyzed with consideration of anemia and hypoproteinemia, and various complications within the whole period of hospital stay.

First of all, the dependence of laboratory values and all developed complications was studied in the whole group of patients. As result, the close positive correlation was found between summary amount of complications for each patient and duration of anemia and hypoproteinemia (n = 54, r = 0.598, p = 0.009).

Then the comparative analysis of the main group and the comparison group was conducted in relation to treatment duration, number of complications, and outcomes (the table 5). The findings show that the incidence of complications was 1.8 more seldom in the main group, and the hospital treatment duration was 6.8 days less than in the comparison group. There were not any undesirable effects after introduction of the drug. The cause of death in the main group was abdominal sepsis. Two patients died after multiple organ dysfunctions in the comparison group.

Table 5

Duration of treatment, number of complications and outcomes

Values	Main group	Comparison group	p
Mean hospital stay, (M ± m)	32.8 ± 4.1	39.6 ± 6.6	0.891
Mean number of complications in 1 patient, (M ± m)	0.8 ± 0.2	1.4 ± 0.4	0.353
Deceased (%)	1 (4 %)	2 (7 %)	0.618*

Note: p – reliability of differences according to Mann-Whitney’s test, p* – according to Fisher’s exact test  

There were not any reliable intergroup differences according to the table 5. However, some significant differences were found in amount of complications in each group. Complications did not develop in a half of patients with more mild conditions (51.9 % in the main group, 48.1 % in the comparison group). Patients with more severe complications had 21 complications in the main group, and 39 in the comparison group. One or two complications were found in 10 patients in the main group and in 6 in the comparison group, 3-6 complications – in 2 patients in the main group, and in 7 patients in the comparison group. For further analysis, both groups were divided into subgroups with absence or presence of identified complications.

The table 6 shows the results of comparison of subgroups of patients with complicated course of polytrauma. These subgroups demonstrated higher clinical and laboratory efficiency of the studied group.

Table 6

Structure of complications and duration of anemia and hypoproteinemia in subgroups with complicated course of associated injury

Complications	Subgroups of patients		р
	Main group (n = 13)	Comparison group (n = 13)
All complications, in 1 patient on average	1.6 ± 0.3	3.0 ± 0.4	0.014
– life-threatening infectious complications (severe sepsis, septic shock)	0.8 ± 0.2	0.7 ± 0.3	0.579
– life-threatening non-infectious complications (acute bleeding, arterial thrombosis, pulmonary embolism, lung atelectasis with respiratory failure, fat embolism)	0.1 ± 0.1	0.3 ± 0.2	0.511
- non-life threatening infectious complications (sepsis, focal pneumonia, bronchitis, pleuritis, wound purulence, cystitis, pyelonephritis)	0 ± 0	0.9 ± 0.3	0.044
- non-life threatening non-infectious complications (acute gastrointestinal ulcer, venous thrombosis)	0.7 ± 0.2	1.2 ± 0.2	0.186
Blood hemoglobin < 100 g/l, days	7.1 ± 2.1	16.0 ± 3.1	0.026
Blood hemoglobin < 90 g/l, days	3.2 ± 1.3	7.9 ± 2.1	0.044
Total serum protein < 60 g/l, days	8.2 ± 2.2	19.5 ± 4.1	0.034
Total serum protein < 50 g/l, days	1.1 ± 0.5	5.4 ± 1.6	0.009

Note: p – reliability of differences according to Mann-Whitney’s test.

The table 6 shows the lower (1.9 times) amount of complications in the main group as compared to the control group. It was mainly determined by a decrease in number of infectious, non-life-threatening complications.

The analysis of the laboratory values of erythropoiesis and total protein identified a more favorable correcting effect in the subgroup with sodium deoxyribonucleate.

The duration of acute anemia and hypoproteinemia in all analyzed ranges of hemoglobin and total protein levels was reliably higher in the subgroup with complications of the comparison group (2.3-4.9 times) as compared to patients of the similar subgroup of the main group.

Certainly, for critical states, the significant importance is related to condition of immune system, i.e. a degree of mobilization of neutrophils and monocytes providing the immediate anti-infection protection, and regulation of inflammatory and reparatory processes. For this reason, the patients with severe associated injury were analyzed for blood levels of mononucleatyc cells expressing the receptor of stem cell factor (CD117⁺) as the value of activation of bone marrow hematopoesis and mobilization of leukocytes-precursors and tissue cells into peripheral tissues. The amount of def⁺NG was estimated for influence of sodium deoxyribonucleate on bactericidal properties of leukocytes. It is known that alfa-defensins show the evident cytotoxic activity in relation to various agents, and promote the regulation of cortisol level and reparation of damaged tissues [14].

According to the table 7, the patients of the main group showed an increase in blood monocytes and CD117⁺ in 7 days after introduction of the drug. The increase differed from the levels in the control group. One should consider it as activating influence of the drug on migration of stem cells from bone marrow into the blood flow and, subsequently, into tissues, providing the reparative processes in the injury site.

Table 7

Levels of monocytes, mononuclear cells CD117⁺and def⁺NG in patients with associated injury 

Values	Groups of patients	Before injection	after 7 days	after 14 days	P1,2	P1,3
		1	2	3
Monocytes, х109l	Main group Comparison group	0.56 ± 0.05 0.54 ± 0.06 p = 0.799	0.89 ± 0.07 0.69 ± 0.07 p = 0.049	0.65 ± 0.07 0.46 ± 0.05 p = 0.033	0.004 0.160	0.681 0.526
Mononuclear cells, CD117+, х109l	Main group Comparison group	0.46 ± 0.05 0.44 ± 0.08 p = 0.833	0.81 ± 0.07 0.44 ± 0.07 p = 0.000	0.46 ± 0.05 0.39 ± 0.07 p = 0.421	0.014 0.362	0.237 0.398
Def+NG, %	Main group Comparison group	36 ± 4 41 ± 4 p = 0.382	38 ± 4 37 ± 3 p = 0.842	46 ± 3 33 ± 4 p = 0.044	0.548 0.600	0.037 0.165

Note: p – reliability of differences according to Mann-Whitney’s test, P – according to Wilcoxon’s test

 Moreover, the activating influence of sodium deoxyribonucleate on maturation of functionally mature granulocytes in bone marrow (def⁺NG), which contain antimicrobial and regulatory peptides, and on migration into the blood. It is testified by gradual increase in proportion of def⁺NG in the whole population of blood granulocytes in the main group, achieving 26 % of increase in comparison with the basic level by the day 14 of the follow-up. The patients of the comparison group demonstrated a gradual decrease by 59 % in their proportion among granulocytes (the table 7).

Besides of deficiency of proteins, fats, carbonhydrates, aminoacids and microelements, the disadvantage of exogenous bioavailable nucleotides can influence on severity of the disease course. So, exogenic introduction of native fragments of DNA significantly improves the activity of bone marrow in radial disease, and for oncology – after chemotherapy courses [9]. In the post-shock period of severe associated injury, one can observe the insufficient activity of functioning of bone marrow and peripheral immune system [1].

From the first hours after trauma, patients with associated injury show the high requirement for synthesis of protein and its preservation in the vascular bed, for recovery and maintenance of higher blood hemoglobin to exclude the effects of hypoxia and to maintain the sufficient amount of functionally mature immune cells for realization of antimicrobial protection and recovery of injured tissues. Insufficient reserves for restoration of above-mentioned disorders create some premises for development of various complications, which influence on quality and duration of treatment

 

CONCLUSION

The use of sodium deoxyribonucleate for patients with severe associated injury promotes the recovery of total protein and hemoglobin, increasing migration of stem cells and NG with antimicrobial peptides into the blood. Patients with complicated course of associated injury were more sensitive to the drug. Eventually, 1.8-fold decrease in amount of complications, and the decrease in treatment by 6.8 days were achieved.

As result of the double blinded study, we can state that commercial sodium deoxyribonucleate (Derinat) increases the efficiency of complex treatment of patients with severe associated injury.

The novelty of the study has been protected with application for the invention (acknowledgement of receipt from December 25, 2018).

 

Information on financing and conflict of interests

The study was conducted without sponsorship. The authors declare the absence of any clear or potential conflicts of interests relating to publication of this article.