| Amphotericin B?AmB?is a polyene antibiotic with broad spectrum antifungal activity and used for serious fungal infections.It is typically given by injection into a vein.However,amphotericin B is well known for its severe and potentially lethal side effects and it even at therapeutic doses has also been associated with multiple organ damage.Kidney damage is a frequently reported side effect,and can be severe and/or irreversible.In order to improve the tolerability of amphotericin and reduce toxicity,several lipid formulations have been developed.Liposomal amphotericin B?L-AmB?has been found to have less renal toxicity and fewer infusion-related reactions and has been used for nearly 20 years to treat a wide range of fungal infections.The antifungal activity of amphotericin B is retained after its incorporation into the liposome bilayer,but its toxicity is significantly reduced.We recently noticed that an L-AmB preparation produced more severe toxic reactions in female rats than male rats.So far,there is no report about the phenomenon and thus little is known about the gender differences of acute toxicity for L-AmB preparation.Therefore,this study was designed to further verify gender differences of acute toxicity in the male and female rats by using two L-AmB preparations from different provider.The pharmacokinetic characteristics of L-AmB in male and female rats were studied by HPLC-MS in biological samples,with a view to elucidate the gender differences in acute toxicity.Part1 Acute Toxicity Test of Amphotericin B Liposome in Male and Female Rats Aim:1 To observe gender differences of acute toxic reactions in female and male rats after administration of two L-AmB preparations?No.1 and 2?.2 To observe gender differences of acute toxic reactions in female and male rats after administration of regular AmB preparation.Method:1 Acute toxicity of L-AmB-1: SD rats were randomly divided into four groups and were received a single intravenous dose of L-AmB-1 at 0.5,1.0,2.0 and 4.0 mg/kg,respectively.The responses and mortality of female and male rats were separately observed and recorded.For L-AmB-2,SD rats were randomly divided into two groups and were received a single intravenous dose of L-AmB-2 at 2.0 and 4.0 mg/kg,and each group with 6 male and females.The responses and mortality of female and male rats were separately observed and recorded.2 Acute toxicity of AmB: Forty SD rats were randomly divided into four dose groups including 0.5,1.0,2.0 and 4.0 mg/kg,with 10 rats in each group?half male and half female?.The responses and mortality of female and male rats were separately observed and recorded after the administration of a single intravenous dose of AmB.Results1 Acute Toxicity of L-AmB: After single intravenous injection of L-AmB-1,no toxic symptoms were observed in male and female rats in the group of 0.5mg/kg and toxic responses were observed at dose of 1 mg/kg including convulsions,proneness,dyspnea at 20 to 30 minutes after dosing and gradually diminished within 60 minutes.At 2 mg/kg,rats showed the severe symptoms,and 3 of 15 test female rats died within 2 to 4 hours.But,there was no death in male rats.At 4 mg/kg,11 of 15 female tested animals died within0.5 to 1 h after dose and 5 of 15 male tested animals died within 0.5 to 2 hours.Mortality in female rats was significantly higher than it in male rats.After single intravenous injection of L-AmB-2 at 2 mg/kg,mild toxic reactions were observed in male than female rats.At dose of 4 mg/kg,rats showed severe toxic reactions and 4 of 6 female,and 2 of 6 male animals died.Mortality in female rats was significantly higher than it in male rats.2 Acute Toxicity of AmB: The signs of toxicity were evident after thesingle intravenous of AmB at dose of 0.5 mg/kg both in male and female rats.At dose of 1 mg/kg,rats showed severe toxic reactions and 3 of both 5 female and male tested animals died.At doses of 2 and 4 mg/kg,all of the tested animals including female and male died within 0.5 to 2 hours after the administration.There was no difference in mortality for female and male rats.Summary:1 L-AmB from either of provider showed significant gender differences in acute toxicity after single intravenous administration.Mortality in female rats was significantly higher than it in male rats.Based on the toxic reactions of animals,we deduced that acute toxicity target organ of L-AmB was the central nervous system.2 There was no gender difference in acute toxicity of single intravenous injection of AmB.Part 2 Toxicokinetics of L-AmB-1 in male and female rats Aim:Establishment of HPLC-MS method for the determination of L-AmB-1in blood and tissue and by using this method toxicokinetic of L-AmB-1 and distribution in plasma and tissue of AmB were studies so as to provide explanation for the gender differences in acute toxicity.Method:1 HPLC-MS analysis for AmB: The concentrations of AmB in plasma and tissue samples were analyzed by HPLC-MS assay.The method was validated by testing parameters including specificity,detection limit and quantization limit,matrix effect,precision and accuracy,extraction rate and recovery rate.2 Protocol for toxicokinetic studies: Forty-eight SD rats were randomly divided into three groups of 1,2 and 4 mg/kg,with 16 rats each group and half male and half female.Blood samples were taken at 0.08,0.16,0.25,0.5,1.0,2.0,4.0,8.0,24 and 48 hours after administration of single dose L-AmB-1at 1mL/1kg body weight,and plasma was separated and stored at-20°C.The concentration of AmB in the plasma was determined by HPLC-MS.3 Tissue distribution: Thirty-six SD rats were randomly divided into 3groups,with 12 rats each group and half male and half female.Three groups of rats were intravenously injected L-AmB-1 at a dose of 2 mg/kg.Animals were respectively anesthetized at 0.5,1.0 and 2.0 hours after administration and the heart,liver,kidney,brain tissue were taken and washed with saline blood,and dried by filter paper.Tissues were then weighed and stored at-20 ? for the determination of amphotericin B by HPLC-MS method.Results:1 HPLC-MS method validation: HPLC-MS method for toxicokinetic analysis of AmB in plasma and tissue was successfully established and validated.2 Plasma concentration: The concentrations of AmB in plasma at the dose of 1 mg/kg was fitted for a two-compartment model and non-compartmental models at doses of 2 and 4 mg/kg.The AUC and t1/2 in female tested animals were significantly higher than those in the male,while the female animals showed a significant smaller CL than the male.3 Tissue distribution: The highest concentration of drug are distributed in the liver and kidneys,in which female rats displayed a significant higher concentration than that of male.The concentration of drug in the brain increased with time after administration and was 10.05,18.43,30.06mg×h×mL-1,respectively,wherease,in male was 9.16,13.96,19.58 mg×h×mL-1.Obviously,the drug distribution in brain was higher in females than in males.Summary: The toxicokinetic studies showed that female animals had higher drug exposure,longer half-life and lower plasma clearance compared to male rats.The tissue distribution indicated that the drug was most distributed in the liver and kidneys,in which female rats displayed a significant higher concentration than that of male rats.In addition,the drug distribution in brain was higher in females than in males.Conclusion: There was a significant gender difference for acute toxicity after a single intravenous injection of Liposomal amphotericin B in rats,that is,Mortality in female rats was significantly higher than it in male rats.Toxicokinetics results showed that this gender difference was due to a slowerclearance of drugin female rats,resulting in increased exposure to the body and higher tissue distribution including the brain,which was the toxic target organ in acute toxicity. |
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