Influence of drugs with hyaluronidase activity on the survival of animals with a purulent-inflammatory process in the abdominal organs

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BACKGROUND

Intraabdominal infections remain crucial in clinical medicine because of the high probability of fatal complications [1–3]. Antibacterial therapy optimization is significant in increasing the efficiency of their treatment [4–6]. To achieve this goal, antibacterial drugs should be administered using the lymphotropic method. It involves the injection of a low-molecular-weight hydrophilic drug, particularly a beta-lactam antibiotic, following the administration of a drug with endolymphatic conductor properties. The latter includes ensuring targeted delivery of the antibacterial drug to the lesion through the lymphatic system. In this regard, high-molecular-weight medicinal substances can be used to stimulate lymphatic drainage and maintain high concentrations in the blood plasma of drugs administered after them through the same needle throughout the day, such as hyaluronidase (HLRD) [7, 8]. Previous studies using an experimental model established that in addition to HLRD, such properties are also exhibited by the Russian drug bovhyaluronidase azoximer (BHLRD+Az), which is a conjugate of the HLRD enzyme and a high-molecular-carrier azoximer, which is considered an effective stabilizer that promotes long-term preservation of the native structure and activity of the enzyme [9].

The efficacy of lymphotropic therapy using HLRD as an endolymphatic conductor has been demonstrated in experimental studies [10, 11] and in numerous clinical cases. This treatment method is used in surgery [12–15], gynecology [16], urology [17], neurology [18], pulmonology [19], otolaryngology [20], ophthalmology [21], and other fields of medicine. The treatment efficiency in these studies was assessed using the dynamics of the disease clinical presentation and instrumental and laboratory parameters. To clarify the mechanism of the positive effect of lymphotropic therapy on the course of the intraabdominal infectious process, we conducted a study of the survival of animals with a simulated purulent inflammatory process of the abdominal organs with the combined application of HLRD and BHLRD+Az with the antibacterial drug cefotaxime (CF). We believe that the results obtained experimentally will be of interest in practical medicine.

This study aimed to investigate in experiments on mice the effect of coadministration of cefotaxime with hyaluronidase or bovhyaluronidase azoximer on the survival of animals with abdominal purulent inflammation.

METHODS

The survival rate of 112 mature male mice of the CBA (luc) line with simulated abdominal purulent inflammation was assessed following the combined use of CF and HLRD or BHLRD+Az. The mice weighed 28–30 g, were kept on a standard diet, and had no external signs of any diseases. The dose of all studied drugs was calculated based on a single dose recommended for humans, using a conversion factor [22]. A 0.9% NaCl solution was used as the solvent for the studied drugs. Abdominal purulent inflammation was modeled by intraperitoneal injection of a lethal dose of microbial suspension of S. aureus N: 25923 (J49) ATCC (NSDA, USA). LD₁₀₀ was determined in a series of preliminary experiments and amounted to 10⁹ mg/ml.

Four groups of 28 animals each were formed. Group 1 (control) received no treatment. Group 2 mice were subcutaneously injected with 0.3 ml of 0.9% NaCl in the upper third of the thigh 60 minutes after the administration of the microbial suspension, and then 3 minutes later, CF was injected through the same needle at a dose of 5.9 mg. Groups 3 and 4 mice were subcutaneously injected with HLRD at a dose of 0.26 IU and BHLRD+Az at a dose of 12 IU, respectively, in the upper third of the thigh, and then 3 min later, CF was injected through the same needle at a dose of 5.9 mg.

Statistical analysis. When analyzing the survival of experimental animals, the Kaplan–Meier method of multiplication estimates and univariate analysis using the log-rank test were used to identify significant differences between the compared groups of mice.

RESULTS

Figures 1–3 show Kaplan–Meier curves demonstrating the dynamics of animal survival in the control and experimental groups.

 

Fig. 1. Survival of mice in groups of animals with simulated abdominal purulent inflammation without treatment and those injected with cefotaxime (CF) alone.

 

Fig. 2. Survival of mice in groups of animals with simulated abdominal purulent inflammation injected with cefotaxime (CF) alone and combined administration with hyaluronidase (HLRD).

 

Fig. 3. Survival of mice in groups of animals with simulated abdominal purulent inflammation injected with cefotaxime alone (CF) and combined administration with bovhyaluronidase azoximer (BHLRD+Az).

 

In the group of mice that were not treated (control), all animals died by day 7 of follow-up. In group 2 (experimental), after a single injection of CF at this follow-up period, 11 of 28 mice (39%) survived. The resulting difference was statistically significant (95% CI, 1.119–2.214; p=0.0007). The survival rate of animals treated with CF compared with that of the control group increased from the first days of follow-up. Thus, on day 2, it was 82% in the experimental group (CF) and 68% in the control group. On days 3, 4, and 5, survival rates were 68% and 46%, 51% and 32%, and 43% and 10%, respectively. By day 7, no animal in the control group survived, whereas 43% of the mice that received a single dose of CF survived (Fig. 1).

To assess the effect of the studied drugs on the survival of mice with a simulated purulent inflammatory process of the abdominal organs, we compared this indicator in experimental groups of animals with combined administration of CF and HLRD or CF and BHLRD+Az and in a group of mice that received CF as a single drug. Moreover, in this context, animals with CF single administration were considered as the control group. It was revealed that both HLRD and BHLRD+Az increased the survival rate of the experimental animals.

As previously revealed by day 7 of follow-up, 11 animals (39%) survived in the group of mice that received a single injection of CF. When CF was combined with HLRD, 21 of 28 (75%) mice remained alive. The 95% CI was 0.117–0.642 (p=0.0029). Differences in the survival of animals in these study groups were noted starting from day 2 of the experiment, as this figure was 96% in the main group and 82% in the control group. On days 3 and 4, it was 93% and 68% and 93% and 57%, respectively. On days 5 and 6 of the experiment, the survival rate of animals in the group with combined administration of CF and HLRD was 86%, and in animals that received the antibiotic as a single drug, it was 43% (Fig. 2).

In the group of mice with combined administration of CF and BHLRD+Az, 20 animals (71%) survived on day 7 of the experiment, whereas in the control (monoantibiotic administration), there were 11 (39%) such animals. The data obtained were statistically significant (95% CI, 0.171–0.897; p=0.0027). Differences in the survival rate of mice in these groups were recorded starting from day 3 of follow-up. Thus, the survival rate on day 3 was 79% in the main group of animals and 68% in the control group, and it was 71% and 57% on day 4, respectively. On days 5–6, it was 71% in mice treated with CF combined with BHLRD+Az and 43% in animals that received CF alone (Fig. 3).

DISCUSSION

A single injection of CF, both as monotherapy and against the administration of the studied drugs, ensured the survival of some mice with simulated abdominal purulent inflammation compared with the control group (without treatment), where all the animals died by the end of the experiment (day 7). Moreover, the best results were achieved in animals with preliminary administration of HLRD or BHLRD+Az. Thus, on day 7 of follow-up, >70% of the animals survived in the groups of mice with combined administration of the antibiotic and HLRD or BHLRD+Az. Statistically significant differences in the effect of these two drugs on the survival of mice under conditions of an experimental purulent inflammation have not been detected.

The literature presents evidence that the combined administration of recombinant HLRD with certain drugs improves the delivery of small molecules into the blood and increases their bioavailability and maximum concentration (and reduces the time to reach it) compared with subcutaneous injection of drugs without HLRD [23]. However, the use of HLRD in lymphotropic therapy can cause allergic reactions, whereas they are very rare when using BHLRD+Az [24].

We previously established that HLRD and BHLRD+Az accelerate lymphatic drainage in the mesentery of experimental mice [25, 26]. Preliminary administration of these drugs increases the concentration of CF throughout the day in both the blood and intestinal tissues. This effect may be due to the presence of endolymphatic conductors in HLRD and BHLRD+Az (endolymphatic conductors deliver drugs to the lymphatic system and deposit them there with gradual release into the blood). This mechanism may determine the effect of increasing the survival rate of animals with abdominal purulent inflammation.

CONCLUSION

The results obtained enable the use of HLRD and BHLRD+Az for lymphotropic therapy. In this case, BHLRD+Az should be preferred, because its use is rarely accompanied by allergic reactions.

Additional Information

Funding source. This study was not supported by any external sources of funding.

Competing interests. The authors declare that they have no competing interests.

Authors' contribution: G.V. Kukushkin — idea, research design, set of the material, manuscript writing and editing; M.V. Zhuravleva — manuscript writing and editing; D.E. Yurov — set of the material, search for literary sources. All authors made a substantial contribution to the conception of the work, acquisition, analysis, interpretation of data for the work, drafting and revising the work, final approval of the version to be published and agree to be accountable for all aspects of the work.

About the authors

German V. Kukushkin

Pirogov Russian National Research Medical University

Email: germanpharm@yandex.ru
ORCID iD: 0000-0002-1661-1071

MD, Cand. Sci. (Med.), associate professor

Russian Federation, 1 Ostrovityanova street, 117997 Moscow

Marina V. Zhuravleva

I.M. Sechenov First Moscow State Medical University (Sechenov University); Scientific Centre for Expert Evaluation of Medicinal Products

Email: clinpharm23@mail.ru
ORCID iD: 0000-0002-9198-8661
SPIN-code: 6267-9901

MD, Dr. Sci. (Med.), professor

Russian Federation, 1 Ostrovityanova street, 117997 Moscow; Moscow

Dmitry E. Yurov

Pirogov Russian National Research Medical University

Author for correspondence.
Email: dmpharm@gmail.com
ORCID iD: 0000-0003-0178-8736
SPIN-code: 6403-4087

MD, Cand. Sci. (Med.)

Russian Federation, 1 Ostrovityanova street, 117997 Moscow

References

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