帕拉米韦原料药的稳定性研究

目的考察帕拉米韦原料药在不同条件下的稳定性。方法采用高效液相色谱法。Welch ODS C18色谱柱(250mm×4.6 mm,5μm);流动相水(含0.1%冰醋酸)–乙腈(90∶10);检测波长210 nm;体积流量1.0 m L/min;柱温:25℃;进样量10μL。考察了帕拉米韦原料药在不同温度、光照和不同p H值下的稳定性。结果帕拉米韦原料药在100℃条件下48 h内是稳定的;在光照10 d的情况下,帕拉米韦的稳定性良好;帕拉米韦在碱性和中性环境下稳定性良好,在酸性条件下不稳定,随着p H值的降低,稳定性越差,在p H 1.0时,降解半衰期(t1/2)是43.6 h。结论帕拉米韦在酸性环境中极不稳定,在碱性和中性条件下稳定性良好。     

帕拉米韦三水合物细菌内毒素检查法的研究

目的：研究并建立原料药帕拉米韦三水合物的细菌内毒素（凝胶法）检查法。方法：取批量生产的,连续批号的原料药帕拉米韦三水合物,参考该原料制剂注射液的用法用量,计算出该原料药的细菌内毒素限值;制备不同浓度的系列溶液;应用两个不同生产厂家的鲎试剂,进行凝胶法干扰预试验和干扰试验。之后,依据确定的限值及最大不干扰浓度,取不同批号的供试品进行细菌内毒素检查。结果：原料药帕拉米韦三水合物在浓度为0.5 mg·mL-1时对鲎试剂与细菌内毒素的凝集反应无干扰作用,细菌内毒素限值可设定为0.5 EU·mg-1。结论：帕拉米韦三水合物可采用细菌内毒素检查法（凝胶法）作为质量控制方法,该方法简便可行。     

高效液相色谱法测定不同时间人血浆中帕拉米韦的浓度及临床观察

目的 测定帕拉米韦给药后不同时间人血浆中的药物浓度,观察病人用药后安全性及疗效,为临床使用帕拉米韦提供依据.方法 高效液相色谱(HPLC)法测定帕拉米韦的血药浓度,以5 mmol/L磷酸二氢钾溶液(0.1％三乙胺)-乙腈为流动相,磷酸调pH 5.0;检测波长210 nm;流速1.0 mL/min;用药后对病人进行安全性随访.结果 血浆中无干扰测定的内源性物质,线性范围为1～50μg/mL,最低定量限(LLOQ)为1μg/mL,回收率102.5％～110.4％,日间精密度及稳定性均良好,5例病人不同时间血浆中的帕拉米韦均可检出;5例病人的发热缓解时间分别为33、26、26、48、54 h,流感症状缓解时间分别为89、>120、28、48、54 h,发生1例不良反应.结论 HPLC检测方法可靠,适用于人血浆中帕拉米韦的测定.帕拉米韦能快速缓解流感症状,虽然出现的不良事件为轻度,但也要监测其不良反应,以保证用药安全性及有效性.     

帕拉米韦原料含量测定方法比较研究

目的：建立并比较高氯酸非水滴定法和高效液相色谱法测定帕拉米韦原料药的含量。方法：非水滴定法：以无水冰醋酸为溶剂,0.1 mol/L的高氯酸滴定液为滴定剂,电位法指示滴定终点进行滴定。高效液相色谱法：采用Welch Ultimate AQ-C₁₈(150 mm×4.6 mm,5μm)色谱柱,以1 mmol/L磷酸二氢钾缓冲液(称取磷酸二氢钾约0.136 g溶于1 000 ml水,加入1 ml三乙胺,并以磷酸调pH 6.0)为流动相A,以乙腈为流动相B,90%A相等度洗脱10 min。流速为1.0 ml/min,柱温为40℃,检测器波长为210 nm,进样量为20μl。结果：两种方法均符合方法学验证要求。对同一批次样品进行测定,结果无显著性差异(P<0.05)。结论：两种方法均能用于帕拉米韦含量测定,为其质量控制提供依据,且各有优势。可根据实际情况和需求选择相应的方法。     

帕拉米韦巯基二肽前药的合成及其稳定性研究

目的合成一系列帕拉米韦巯基二肽前药并考察其在不同介质中的稳定性。方法以帕拉米韦结构中1位羧基为反应位点,巯基二肽为载体,通过酯化反应合成一系列帕拉米韦巯基二肽前药。采用高效液相色谱法测定帕拉米韦巯基二肽前药在不同pH值缓冲溶液、大鼠胃液、大鼠小肠液、人工胃液(含胃蛋白酶)、人工肠液(含胰酶)、大鼠血浆中的稳定性,采用一级动力学方程计算降解速率常数。结果合成了4个帕拉米韦巯基二肽前药,它们在胃肠道中稳定性良好。结论帕拉米韦巯基二肽前药具有良好的稳定性,为帕拉米韦口服给药的开发可提供依据。     

HPLC法测定帕拉米韦的含量及其有关物质

建立帕拉米韦含量及有关物质的HPLC测定方法。色谱柱:Agilent Zorbax SB C18(250 mm×4.6 mm,5μm);流动相:含0.1%三乙胺的0.005 mol·L-1磷酸二氢钾溶液(pH 6.0)-乙腈(93∶7);检测波长:210 nm。     

神经氨酸酶抑制剂帕拉米韦的合成及其抗禽流感病毒药效

目的 改进神经氨酸酶抑制剂帕拉米韦合成方法,提高其反应收率,确定其对人工感染禽流感病毒小鼠的防治效果、适宜剂量及给药方式.方法 以文斯内酰胺和2-乙基丁醛为主要原料,经催化开环、氨基保护、环化加成等步骤合成了帕拉米韦,通过1H NMR,13C NMR及MS等方法确定了中间体及目标化合物的结构.考察不同剂量和给药方式的帕拉米韦预防及治疗人工感染H9N2亚型禽流感病毒小鼠的药效.结果 合成帕拉米韦的总收率为52.7%,较文献提高了 28%.帕拉米韦对感染小鼠的保护效果具有剂量和治疗时间依赖性.40 mg/kg剂量的帕拉米韦一次性肌注,对感染小鼠的保护率为100%.感染病毒后2d内给药疗效较好.结论 帕拉米韦对感染禽流感病毒小鼠具有显著的防治效果,肌注效果较好.     

帕拉米韦治疗流行性感冒的疗效及安全性分析

目的:帕拉米韦是新型的抗病毒药物,本研究拟评价帕拉米韦治疗流行性感冒的临床安全性。方法:以北京大学第三医院感染疾病科发热门诊就诊的流行性感冒患者89例为研究对象,所有患者均给予帕拉米韦氯化钠注射液3 mg·mL~(-1)治疗,单次静脉滴注,评价其发热、呼吸道和全身症状的缓解情况和药物的安全性。结果:患者平均年龄(28.31±8.30)岁。诊断为甲型流感84例,乙型流感5例。治疗后7 d随访,发热均缓解,缓解时间(24.92±14.44) h。主要不良事件包括消化道症状5例(5.62%),神经系统症状3例(3.37%)。心电图由治疗前的窦性心律变为治疗后的窦性心动过缓9例(10.11%),窦性心律不齐6例(6.74%),2例治疗后QTc间期延长。治疗前正常,治疗后2例丙氨酸氨基转移酶异常,15例(16.85%)甘油三酯升高,6例(6.74%)网织红细胞异常。无严重不良事件。结论:帕拉米韦治疗流行性感冒的安全性相对较高。     

帕拉米韦及其中间体酯化物结构的X射线衍射分析

目的 验证所合成的化合物为目标产物——帕拉米韦中间体酯化物及帕拉米韦成品,并通过X射线衍射技术探讨化合物晶型.方法 选择适宜的条件,培养出适合单晶X射线衍射分析的单晶体,对所得单晶数据进行结构解析,得到化合物的三维空间结构信息,与文献进行比对;同时通过单晶结构数据模拟获得化合物的粉末X射线衍射理论图谱,用于晶型研究.结果 单晶X射线衍射结构分析结果表明,帕拉米韦中间体酯化物及成品的化学结构、构型与文献报道完全一致:粉末X射线衍射理论图谱可作为晶型对照图谱使用,并进一步表明中间体存在多晶型现象.结论 单晶X射线衍射分析法可准确测定含多手性中心的帕拉米韦中间体酯化物及成品的空间结构,充分证实了该药物合成过程与结果的正确性;粉末X射线衍射理论图谱可为化合物晶型研究提供有力支持.     

UPLC-MS/MS法测定帕拉米韦在大鼠体内的药物动力学

目的建立测定帕拉米韦在大鼠血浆中药物浓度的UPLC-MS/MS的方法,并且将这种方法用于帕拉米韦口服和静脉注射后大鼠体内的药动学。方法色谱柱为C18柱(50 mm×2.1 mm,1.7μm),使用梯度洗脱来分离被检测的物质,通过沉淀蛋白方法处理被测的血浆样品,采用多反应监测(multiple reaction monitoring,MRM)的方法对被测的样品进行扫描和检测。结果在线性范围内,帕拉米韦的线性关系良好,准确度为-8.0%~+8.0%之间,日内RSD<7.1%且日间RSD<7.1%,平均提取回收率>85%,基质效应为90%~110%。结论该方法简单易操作,适合于帕拉米韦在大鼠体内的药动学研究。     

在体单向肠灌流模型研究帕拉米韦拟肽类衍生物的大鼠小肠吸收特性

目的研究帕拉米韦及其拟肽类衍生物的大鼠小肠吸收机制,筛选出膜渗透性最大的衍生物。方法采用大鼠在体单向灌流法研究帕拉米韦拟肽类衍生物的小肠吸收,采用高效液相色谱法测定药物和酚红的浓度。建立lgD预测值和lgP之间的关系。结果帕拉米韦拟肽类衍生物的膜渗透系数都比帕拉米韦高,其中帕拉米韦L-异亮氨酸衍生物具有最高的膜渗透性;寡肽转运蛋白(PEPT1)典型底物甘氨酰肌氨酸能显著降低帕拉米韦拟肽类衍生物的小肠吸收,而L-缬氨酸不具有这种能力。结论帕拉米韦拟肽类衍生物是PEPT1的底物,它们在大鼠小肠内的吸收是PEPT1介导的主动转运过程。     

帕拉米韦氯化钠注射液治疗儿童流感病毒感染的临床疗效和安全性对比

目的：探讨帕拉米韦氯化钠注射液治疗儿童流感病毒感染的临床效果和安全性与磷酸奥司他韦、喜炎平的回顾性对比研究。方法：选取2015年11月-2016年12月某院发热门诊诊治的1 358例儿童流感病例,根据筛选符合入组条件者1 350例并随机分为A、B、C 3组,每组450例。A组采用帕拉米韦氯化钠注射液治疗,B组采用磷酸奥司他韦颗粒治疗,C组采用喜炎平治疗,比较3组临床疗效和安全性。结果：A组治疗总有效率为92.2%,显著高于B、C组,差异有显著性（P<0.05）;A组体温恢复正常时间、咳嗽、咽痛、鼻塞、流涕缓解时间,显著短于B、C组,差异有显著性（P<0.05）;药物不良反应以消化道症状和化验检查为主,A组发生率为26%,与B（35.1%）、C组（30.2%）比较,差异无显著性（P>0.05）。结论：儿童流感病例采用帕拉米韦氯化钠注射液治疗效果理想,较奥司他韦、喜炎平更快地改善发热和流感症状,可单次静脉注射治疗,安全性高,患儿依从性好,能提高临床治疗效果,值得推广应用。     

黄芩苷联合帕拉米韦体内外抗甲型H1N1流感病毒作用

目的评价黄芩苷与帕拉米韦联合用药体内外抗甲型H1N1流感病毒作用。方法体外试验中,以甲型H1N1流感病毒感染MDCK细胞,黄芩苷与帕拉米韦联合用药,终点稀释法检测细胞上清液病毒滴度;体内试验中,以甲型H1N1流感病毒感染BALB/c小鼠,黄芩苷灌胃,帕拉米韦肌肉注射,两者联合给药,观察试验小鼠存活情况及体重变化。试验结果以MacSynergyⅡ软件分析两种药物体内外联合作用结果。结果细胞试验中,黄芩苷与帕拉米韦联用抗甲型H1N1流感病毒在95%置信区间内的协同值为3.2,表现为相加作用;小鼠试验中,黄芩苷与帕拉米韦联用,对提高感染流感病毒小鼠的存活率和抑制其体重下降表现为显著协同作用,协同值分别为69.0和105.2。结论黄芩苷与帕拉米韦联合抗流感作用比单独使用效果好,在临床上具有重要的应用价值。     

Clinical efficiency in children treated with intravenous drip infusion of peramivir

The clinical effect of peramivir was examined retrospectively in 30 children aged 23 days to 8 years; they had been treated with peramivir at our hospital within 48 hours after the onset of influenza from November 2010 to April 2011. Intravenous dripping of peramivir at a dose of 10 mg/kg was performed only once for 15 to 30 minutes. To examine the clinical effect of peramivir, the time from dosing to defervescence (body temperature <37.5 degrees C) was evaluated. To compare the clinical effects of peramivir and oseltamivir, the time required for the defervescence was evaluated in a similar manner in 30 children aged 7 months to 8 years who had been treated for influenza with oseltamivir at a dose of 4 mg/kg/day. The influenza type was type A (n=25) and type B (n=5) in the peramivir group and type A (n=26) and type B (n=4) in the oseltamivir group. In the peramivir group, 5 (16.7%), 16 (53.3%), and 24 (80.0%) patients achieved defervescence within 6, 12, and 24 hours, respectively. In the oseltamivir group, 3 (10.0%) and 6 (20.0%) patients had defervescence within 12 and 24 hours, respectively. The difference between these groups was statistically significant (p<0.05). An increase in the levels of AST and ALT was observed as an adverse reaction in 1 patient. Thus, peramivir was determined to be effective to influenza in children.     

Characterization of the binding affinities of peramivir and oseltamivir carboxylate to the neuraminidase enzyme

With the continued threat of morbidity and mortality from influenza and the development of resistance to influenza antiviral drugs, there is increasing interest in new treatments, such as the investigational intravenous drug peramivir, and in combination treatments. In this study, we determined the impact of oseltamivir carboxylate on the binding affinity of peramivir/neuraminidase (NA) enzyme complex and vice versa. Influenza NA was incubated with peramivir and oseltamivir carboxylate alone and in combination. Dissociation rates of the enzyme-inhibitor complex measured in the presence of NA substrate for peramivir alone and the combination were similar, suggesting that peramivir competitively inhibits the neuraminidase enzyme and that oseltamivir carboxylate when added to peramivir does not impact the binding affinity of peramivir to the NA enzyme.     

Combinations of favipiravir and peramivir for the treatment of pandemic influenza A/California/04/2009 (H1N1) virus infections in mice

Favipiravir, an influenza virus RNA polymerase inhibitor, and peramivir, an influenza virus neuraminidase inhibitor, were evaluated alone and in combination against pandemic influenza A/California/04/2009 (H1N1) virus infections in mice. Infected mice were treated twice daily for 5 d starting 4 h after virus challenge. Favipiravir was 40%, 70%, and 100% protective at 20, 40, and 100 mg/kg/d. Peramivir was 30% protective at 0.5 mg/kg/d, but ineffective at lower doses when used as monotherapy. Combinations of favipiravir and peramivir increased the numbers of survivors by 10-50% when the 0.025, 0.05, and 0.1 mg/kg/d doses of peramivir were combined with 20 mg/kg/d favipiravir and when all doses of peramivir were combined with 40 mg/kg/d favipiravir. Three-dimensional analysis of drug interactions using the MacSynergy method indicates strong synergy for these drug combinations. In addition, an increase in lifespan for groups of mice treated with drug combinations, compared to the most effective monotherapy group, was observed for the 0.025, 0.05, and 0.1 mg/kg/d doses of peramivir combined with favipiravir at the 20 mg dose level. Therefore, the 20 mg/kg/d dose of favipiravir was selected for further combination studies. Increased survival was exhibited when this dose was combined with peramivir doses of 0.1, 0.25 and 0.5 mg/kg/d (1 mg/kg/d of peramivir alone was 100% protective in this experiment). Improved body weight relative to either compound alone was evident using 0.25, 0.5, and 1 mg/kg/d of peramivir. Significant reductions in lung hemorrhage score and lung weight were evident on day 6 post-infection. In addition, virus titers were reduced significantly on day 4 post-infection by combination therapy containing favipiravir combined with peramivir at 0.25 and 0.5 mg/kg/d. These data demonstrate that combinations of favipiravir and peramivir perform better than suboptimal doses of each compound alone for the treatment of influenza virus infections in mice.     

Combination of peramivir and rimantadine demonstrate synergistic antiviral effects in sub-lethal influenza A (H3N2) virus mouse model

Efficacy of combination of the intramuscularly administered neuraminidase (NA) inhibitor, peramivir, and the orally administered M2 ion channel blocker, rimantadine was evaluated in mouse influenza A/Victoria/3/75 (H3N2) model. Mice were challenged with a sub-lethal virus dose (0-40% mortality in placebo group) and changes in body weights were analyzed by three-dimensional effect analysis to assess mode of drug interactions. Compounds were administered in a 5-day treatment course starting 1h before viral inoculation. The peramivir and rimantadine doses ranged from 0.3-3 mg/kg/d and 5-30 mg/kg/d, respectively. The maximum mean weight loss of 5.19 g was observed in the vehicle-infected group on day 10. In the 1 and 3 mg/kg/d peramivir monotherapy groups, the weight losses were 4.3 and 3.55 g, respectively. In the rimantadine monotherapy group, the weight losses were 3.43, 2.1, and 1.64 g for the 5, 10, and 30 mg/kg/d groups, respectively. Combination of 1mg/kg/d peramivir with 5 and 10 mg/kg/d rimantadine produced weight losses of 1.69 and 0.69 (p<0.05 vs. vehicle and individual agent), respectively, whereas the combination of 3.0 mg/kg/d peramivir with 10 and 30 mg/kg/d rimantadine did not show any weight loss (p<0.05 vs. vehicle and individual agent). The three-dimensional analysis of the weight loss for the majority of the drug combinations of peramivir and rimantadine tested demonstrated synergistic antiviral effects.     

Anti-influenza virus activity of peramivir in mice with single intramuscular injection

In the event of an influenza outbreak, antivirals including the neuraminidase (NA) inhibitors, peramivir, oseltamivir, and zanamivir may provide valuable benefit when vaccine production is delayed, limited, or cannot be used. Here we demonstrate the efficacy of a single intramuscular injection of peramivir in the mouse influenza model. Peramivir potently inhibits the neuraminidase enzyme N9 from H1N9 virus in vitro with a 50% inhibitory concentration (IC(50)) of 1.3+/-0.4 nM. On-site dissociation studies indicate that peramivir remains tightly bound to N9 NA (t(1/2)>24h), whereas, zanamivir and oseltamivir carboxylate dissociated rapidly from the enzyme (t(1/2)=1.25 h). A single intramuscular injection of peramivir (10mg/kg) significantly reduces weight loss and mortality in mice infected with influenza A/H1N1, while oseltamivir demonstrates no efficacy by the same treatment regimen. This may be due to tight binding of peramivir to the N1 NA enzymes similar to that observed for N9 enzyme. Additional efficacy studies indicate that a single injection of peramivir (2-20mg/kg) was comparable to a q.d.x 5 day course of orally administered oseltamivir (2-20mg/kg/day) in preventing lethality in H3N2 and H1N1 influenza models. A single intramuscular injection of peramivir may successfully treat influenza infections and provide an alternate option to oseltamivir during an influenza outbreak.