Indian Journal of Cancer Medknow Publications on behalf of Indian Cancer Society ISSN: 0019-509X EISSN: 1998-4774 Vol. 43, Num. 1, 2006, pp. 12-15

Indian Journal of Cancer, Vol. 43, No. 1, January-March, 2006, pp. 12-15

Original Article

Comparison of uroprotective efficacy of mesna and amifostine in Cyclophosphamide- induced hemorrhagic cystitis in rats

Kanat Ozkan, Kurt E, Yalcinkaya U, Evrensel T, Manavoglu O

Uludag University, Faculty of Medicine, Department of Medical Oncology, Gorukle 16059, Bursa
Correspondence Address:Uludag University, Faculty of Medicine, Department of Medical Oncology, Gorukle 16059, Bursa, ozkanat@uludag.edu.tr

Code Number: cn06002

Abstract

Keywords: Amifostine, cyclophosphamide, hemorrhagic cystitis, mesna

Cyclophosphamide (CYP) is an alkylating chemotherapeutic agent that is used in a variety of malignant conditions including lymphomas, ovarian and breast cancer. Hemorrhagic cystitis (HC) is a well known and a potentially life-threatening adverse effect of this drug. The incidence of HC is related to the dosage and may be as high as 75% in patients receiving a high intravenous dose.[1]

CYP is a pro-drug that is transformed into the active metabolites phosphoramide mustard and acrolein by the P-450 enzymes in the liver. Acrolein has been proven to be a potent urothelial irritant and is currently accepted as the major cause of CYP-induced HC. It has been proposed that urothelial damage occurs by direct contact with acrolein, which causes edema, ulceration, neovascularization, hemorrhage, and necrosis.[2]

Adequate hydration and the concurrent administration of sodium-2-mercaptoethane (mesna) are the most widely employed methods for prevention of CYP-induced HC in clinical practice. However, HC still occurs in 10-40% of mesna-treated patients.[3] Therefore, there is a need for new effective agents for prevention of this side effect.

Amifostine is a broad-spectrum cytoprotective agent with wide potential for use in cancer therapy.[4],[5],[6] It is a prodrug that is dephosphorylated by the plasma membrane-bound enzyme alkaline phosphatase to its active metabolite, the free thiol WR-1065. WR-1065 is rapidly taken up into cells and is the major cytoprotective metabolite. WR-1065 exerts its protective action by scavenging free radicals, by donating hydrogen ions and depleting oxygen, and by neutralizing DNA-reactive moieties of antineoplastic drugs.[4],[5] In preclinical and clinical studies, amifostine showed selective protection of normal tissues against chemotherapy-induced toxicities without influencing the antitumor activity of cytotoxic therapy.

The cytoprotective potential of amifostine has been established for various tissues including hematopoietic progenitor cells, renal cells, myocardium, intestinal epithelium and neuronal cells.[4] Amifostine can offer a chance of protecting urothelium from toxicity of CYP. Srivastava et al[7] evaluated the role of amifostine in the prevention of CYP-induced HC in the rat model and demonstrated that amifostine protects the urothelium against CYP. However, they did not compare its efficacy with mesna in their study. Thus, in this study, we aimed to investigate efficacy of amifostine in the prevention of CYP-induced HC and compare its efficacy with mesna.

Materials and Methods

A total of 40 male Wistar rats, weighing 150-200 g, were allowed free access to food and water and were randomly assigned to one of the four groups of 10 rats each: Group I: controls; group II: received 200 mg/kg of CYP (Endoxan, IE. Ulagay, Istanbul, Turkey) alone by intraperitoneal (i.p.) injection; group III: received 200 mg/kg of amifostine (Ethyol, Er-Kim, Istanbul, Turkey) i.p., 15 min prior to CYP as above; group IV: received 40 mg/kg mesna (Uromitexan, IE. Ulagay, Istanbul, Turkey) i.p., at the same time and 4 and 8 h after CYP administration. Control animals received sterile saline 0.9% i.p. CYP, mesna and amifostine were dissolved in 0.9% sterile saline.

The experimental protocol was in accordance with the guidelines approved by the Declaration of Helsinki for use of animal experiments. All procedures involving animals were approved by our institution′s animal-care committee.

Induction of HC
CYP (200 mg/kg) was injected i.p. to consistently induce HC, as previously described.[8]

Administration of amifostine
Amifostine (200 mg/kg) was administered i.p., 15 min prior to the administration of CYP. This dose was selected on the basis of previous studies on cytoprotection in rats.[7]

Administration of mesna
Mesna was administered according to classical protocol (% 20 of CYP whole dose at the time of chemotherapy initiation, followed by repeat equivalent doses of mesna every 4 h thereafter, for a total of three doses).

The animals were sacrificed by cervical dislocation 24 h after the administration of CYP. Their bladders were removed by careful dissection and fixed 10% formalin. Data from the literature using CYP-induced HC has shown that the best time to access HC in rats is between 24 and 48 h.[9] Therefore, we chose to access CYP-induced HC at 24 h in this study.

Macroscopic evaluation
Bladders were examined grossly for edema and hemorrhage according to Gray′s criteria.[10] Edema was considered severe (3+) when fluid was seen externally and internally in the bladder walls; moderate (2+) when confined to the internal mucosa; mild (1+) between normal to moderate; and absent (0). Hemorrhage was scored as follows: (3+) intravesical clots; (2+) mucosal hematomas; (1+) telangiectasia or dilatation of the bladder vessels; and (0) normal.

Histopathological evaluation
Bladders were fixed in formalin, embedded in paraffin, and processed for hematoxylin and eosin staining. Histopathological changes were scored as follows: (0), normal epithelium and absence of inflammatory cell infiltration and ulceration; (1+), mild changes involving reduction of epithelial cells, flattening with submucosal edema, mild hemorrhage, and few ulcerations; (2+), severe changes including mucosal erosion, inflammatory cell infiltration, fibrin deposition, hemorrhage, and multiple ulcerations.

The results are expressed as the median (min-max), and P < 0.05 was assessed as statistically significant. All of the numeric data were analyzed first using nonparametric Kruskal-Wallis test to find whether there is difference between groups, and then Mann-Whitney U-test was performed to analyze two groups consecutively.

Results

Discussion

In this study, we investigated the role of amifostine in the prevention of CYP-induced HC and compared its efficacy with mesna. We have confirmed that amifostine could be a useful agent in the preventive management of CYP-induced HC. We have also demonstrated that amifostine was as effective as mesna in this setting. The major disadvantage of our study is the small sample size. However, we assume that this observation provides recent evidence that could modify the prophylactic approaches to CYP-induced bladder damage.

Apart from preventing HC, there could be other advantages of using amifostine in this situation. The use of CYP is associated with mutagenesis and bladder cancers, particularly in those patients who developed cystitis.[11],[12] It is therefore possible that when used in conjunction with CYP, amifostine may not only provide immediate protection from HC, but also prevent mutagenesis and development of secondary malignancies. Furthermore, some studies demonstrated that amifostine could reduce the severity of CYP-induced myelotoxicity and improve patient tolerability to the therapy.[13]

Amifostine was used at a dose of 200 mg/kg in our study. This dose is equivalent to about 1300 mg/m ² in rats. If clinical trials are to be initiated, administration dose of amifostine needs to be considered. The ideal dose of amifostine for different clinical situations remains to be defined, but doses of 740 mg/m ² or 910 mg/m ² have been shown to provide cytoprotection in most studies.

The mechanism of protection by amifostine to HC is a matter of dispute. WR-1065 might provide bladder protection by several mechanisms. WR-1065 can bind directly to acrolein and detoxify it. Therefore, nontoxic thioester does not induce any damage to the urothelium. However, CYP-induced HC is probably not only due to direct contact of acrolein with bladder mucosa. Active oxygen species (AOS) and oxygen free radicals produced by CYP are most likely contribute to occurrence of bladder damage via several mechanisms including peroxidation of membrane lipids, protein denaturation and DNA damage. WR-1065 is a potent scavenger of active oxygen species. This feature of WR-1065 probably mediates the protective effect of amifostine against CYP-induced bladder damage. These hypotheses should be established by further studies focusing on the anti-apoptotic effects of amifostine on urethelium, as previously demonstrated on hematopoietic cells by Mazur et al.[14]

In conclusion, results of this study demonstrate the efficacy of amifostine in the prevention of cyclophosphamide-induced hemorrhagic cystitis. This result is encouraging. However, how this will translate to clinical protection of patients from CYP-induced HC remains to be established.

References

Copyright 2006 - Indian Journal of Cancer

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