| Background and ObjectivesBackground:It has been proved remarkably effective for systemic application of amifostine to prevent tissues from irradiation-induced injuries and it has been used in the clinic. After years of use and research, the drug delivery method has appeared some shortages. For example, it requires a skilled practitioner to administer the dose; the incidence of hypotension is very high and even serious; and it can lead to some side effects which it is difficult to prevent and treat in the clinic. These disadvantages hinder its extensive use. At present, many studies report that intrarectal application of amifostine can significantly reduce the severity of radiation-induced rectal injury and there are many corresponding clinical studies to support those conclusions. For the patients with head and neck cancers, radiotherapy is an important therapeutic measure. However, it can inevitably cause radiation damages. So if this method of administration of amifostine has been proved feasible and favorable, it no doubt by the emergence of this novel usage of amifostine for suffering patients brought good news. Because there are many defects in the existing detection methods of amifostine such as operational complexity and low sensitivity, besides, it is hard to detect directly amifostine and its metabolite WR-1065at the same time and it takes too much time. So it is necessary for us to establish a simpler, faster and more sensitive detection method of amifostine and WR-1065. The study is divided into three parts.Objectives:1. To establish a method of HPLC-MS/MS (High performance liquid chromatography-tandem mass spectrometry) for the direct determination of amifostine in human saliva.2. To preliminarily investigate the feasibility of topical application of the radioprotective compound WR-2721to the buccal mucosa.3. To preliminarily explore the effectiveness of topical application of the radioprotective compound WR-2721to the buccal mucosa.Methods1. Development of the detection method:Saliva samples were collected from6adult healthy volunteers. After appropriate treatment and addition of the internal standard (IS) huperzine-A (HupA), HPLC-MS/MS was first used to analyse amifostine in human saliva by protein precipitation. The analysis was conducted using a ZIC(?)-HILIC analytical column(100×2.1mm,3.5μm). Analysis in mass was operated by the electrospray ionization in the multiple reaction monitoring (MRM) mode.2. Preliminary investigation of feasibility:Saliva samples were collected from5volunteers and were reconstituted in amifostine solutions to final concentrations of4.5×10-3g/1,9×10-3g/1and18×10-3g/1. Measurements of amifostine and WR-1065were taken after mixing at5min,20min,40min,60min,80min and100min. A total of24young-adult guinea pigs were divided randomly into two different dose groups and a blank control group (Group C) as follows:topical administration of amifostine50mg and100mg (Group A and Group B) to each buccal mucosa. Each group was further divided randomly into three subgroups (Omin-subgroup,15min-subgroup and30min-subgroup). Analyses of amifostine and its active metabolite WR-1065were conducted using high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Statistical analysis was carried out with SPSS17.0package.3. Preliminary study of effectiveness:A total of32young-adult guinea pigs were divided randomly into three different groups (A, B and C) and a blank control group (D). There were8guinea pigs in each group. The drug delivery method of amifostine of A、B group was the same to the preliminary investigation of feasibility. The guinea pigs of group C were administered topically normal saline. After giving medicine, the bilateral buccal mucosa of the guinea pigs of group A, B and C were exposed to a single of30Gy X-ray radiation. On the8th day after irradiation, the general conditions of the irradiated guinea pigs, such as weight and behavior were observed. The oral mucositis indices (OMI) of the guinea pigs were measured daily using Parkins scoring system. Histological changes in the irradiated tissues were assayed by H&E staining. Statistical analysis was carried out with SPSS17.0package.Results1. LLOQ (Lowest Limit Of Quantification, LLOQ) of the method was0.938mg·L-1(S/N>10).The standard curve was linear in the range of0.938～30mg·L-1. The linear relationship was good(r=0.9991)(n=6). The relative standard deviations (RSDs) of inter-day and intra-day were all less than15%for the low, medium and high quality control concentration (1.0、5.0and25mg·L-1).The values of recovery were all more than85%.2. The concentrations of amifostine in human saliva in vitro were stable. No WR-1065was detected in saliva. In the guinea pigs from Group A and Group B, there were significant differences in concentrations of amifostine and WR-1065in the tissues between the Omin-subgroup and15min-group and between the Omin-subgroup and the30min-subgroup (P<0.05). The concentrations of amifostine and WR-1065from the15min-subgroup and30min-subgroup did not differ statistically (P>0.05).3. The average scores of group A, B, C and D were respectively2.9,2.4,4.4and0points. There were no significant difference (P>0.05) between group A and Group B. But compared with group C, the damages of the buccal mucosa of the other three groups were relieved remarkably (P<0.05). Histologically, the damages of the tissues and the infiltrating cells were observable in group A, B and C. In group C, scattered ulcerative lesions could be observed with the naked eye. Furthermore, more necrosis and exfoliation of the squamous cells, a large number of infiltrating cells (a majority of which were lymphocytes), and capillary extension and engorgement could be observed by optical microscopy. In group A, a part of inflammatory cells, mild capillary extension and engorgement could be observed. In group B, these pathological changes are slighter than group A. In group D, no inflammatory response could be observed.Conclusions1. The method is direct, fast, simple and sensitive, and suitable for the determination of amifostine in saliva samples and scientific research. It is practicable to detect the concentrations of amifostine and its active metabolite WR-1065in biological samples.2. It is feasible to administer topical amifostine (WR-2721) to mucosa to prevent radiation-induced oral mucositis, and systemic absorption is negligible. Within research time points (5min-100min), relatively high concentrations are maintained in saliva, although some inconsistent changes are observed.3. It is effective for local application of a certain dosage of amifostine to prevent tissues from irradiation-induced injuries. And increase a certain dosage of the drug, though it is not distinct, the pharmacodynamics would be improved. |
PDF Full Text Request