Cyclophosphamide is used for the treatment of lymphoma, leukaemia, some solid  tumours [1] and autoimmune disorders [2]. When carrying out myeloablative cytostatic therapy, the doses of cyclophosphamide are prescribed, which destroy the tumour, but cause fatal pancytopenia [3, 4].

Owing to subsequent allogeneic hematopoietic stem cell transplantation, these doses are an order of magnitude larger than the doses, used for conservative therapy [5], and exceed 120 mg/kg [6]. Early toxic effects of such doses of cyclophosphamide are manifested by asthenic and emetic syndromes [7], limiting the drug tolerance. In rats, bioequivalent doses of cyclophosphamide (≥ 600 mg/kg) caused hyperammonaemia and symptoms, specific to the acute ammonium salt intoxication: ataxia, tremor, loss of reflexes, and seizure [8]. Increased permeability of the intestinal barrier could be the possible mechanism, underlying hyperammonaemia associated with acute cyclophosphamide intoxication [8–11]. Inhibition of intestinal peristalsis increases the permeability of the intestinal wall [12]. Thus, the study was aimed to assess the effects of the myeloablative dose of cyclophosphamide on gastrointestinal peristalsis.

METHODS

The study involved 24 male outbred albino rats with the body weight of 161–190 g, obtained from the Rappolovo laboratory animal nursery. The animals were treated in accordance with the Principles of Good Laboratory Practice, stated in the Order № 199n of the Ministry of Health of the Russian Federation, dated April 1, 2016 [13], and the requirements of the Guidelines for the Housing and Care of Laboratory Animals [14, 15]. Standard rat diet and ad libitum water access were provided. The day before the experiment the rats were deprived of food; however, access to water was not limited.

The rats were randomized into six groups, four animals per group. Water was administered in three control groups (intraperitoneal, subcutaneous, and intragastric routes). The freshly prepared aqueous cyclophosphamide solution in the amount of 10 mL/kg in a dose of 1000 mg/kg was administered in the corresponding three experimental groups. This was an absolutely lethal dose under either route of administration: all animals died within two weeks; in the case of intraperitoneal injection, the dose corresponded to 3.5 LD₅₀.

The 35% aqueous suspension of barium sulfate (10 mL/kg) was administered into the stomach of all rats using the gavage tube immediately after the cyclophosphamide administration. After 1, 3, 5, and 25 h the animals were placed in plastic pencil boxes, and the radiographic testing with the use of the Iconos R200 digital x-ray system (Siemens; Germany) was performed in a pairwise manner (experimental and control animals). X-ray images were assessed using the planimetric ruler by calculating the absolute and relative (percentage) values of the radiopaque shadow area in the stomach, duodenum, jejunum, cecum, descending colon, and rectum. The relative values other than zero or 100%, which were averaged for each group of animals, were ranked and assigned to one of the intervals (1–25, 26–50, 51–75, 76–99%), marked with various shades of grey; no shadow was marked with white, and the shadow of 100% of the injected barium mixture was marked with black. The data obtained were represented as a scheme.

RESULTS

An hour after the barium suspension administration to intact rats, a portion of the suspension passed from the stomach to the duodenum. After 3 h, the suspension was observed in the jejunum, and after 5 h it was also found in the cecum. Twenty-five hours after the start of the experiment the major portion of barium sulfate, found on the x-ray images, was in the descending colon and the rectum.

In the case of intraperitoneal administration of cyclophosphamide, barium suspension never reached the jejunum, and in the case of cyclophosphamide administered intragastrically, the suspension never left the stomach. Subcutaneous cyclophosphamide administration resulted in the less prominent slow-down of transit: after 3 h the radiopaque contrast medium left the stomach, however, after 25 h the medium did not reach the descending colon (fig. 1, fig. 2).

DISCUSSION

The development of gastrointestinal stasis in rats after cyclophosphamide administration is consistent with the earlier reported [16] slowing of gastric motility after the subcutaneous injection of cyclophosphamide in a dose of 50–200 mg/kg to rats. In the case of intragastric administration, the effect size could be due to the aldophosphamide (transport form of cyclophosphamide) hydrolysis in the acidic gastric contents with the formation of more active alkylating metabolites [17, 18].

In our study, the dose of cyclophosphamide corresponded to myeloablative dose for humans of 155 mg/kg [19]. Therefore, the data obtained clearly point to the possibility of developing gastrointestinal stasis in case of using cyclophosphamide to prepare the patients for the allogeneic hematopoietic stem cell transplantation.

Gastrointestinal stasis is a potentially fatal complication found in the intensive care unit patients [20, 21]. The condition increases the permeability of the intestinal barrier [22, 23], and results in the gram-negative bacterial lipopolysaccharides entering the bloodstream, developing systemic inflammation [24, 25] and sepsis [26]. Increased permeability of the intestinal barrier also results in the enhanced flux of toxic nitrogenous metabolites, such as ammonia, from chyme into the bloodstream. In rats, exacerbation of hyperammonaemia after the gavage with ammonium acetate against the background of acute cyclophosphamide intoxication leads to the rapid development of neurological disorders and significantly reduces the animals' life expectancy [27].

CONCLUSION

Modelling myeloablative cytostatic therapy in rats using cyclophosphamide results in gastrointestinal stasis. The changes in gastrointestinal peristalsis reported may contribute to the flux of the gut microbial products into the bloodstream and endotoxemia, and may be involved in the development of the cyclophosphamide early toxic effects.