Metabolism,Excretion, and Mass Balance of the HIV-1 Integrase Inhibitor Dolutegravir in Humans

The pharmacokinetics, metabolism, and excretion of dolutegravir, an unboosted, once-daily human immunodeficiency virus type 1 integrase inhibitor, were studied in healthy male subjects following single oral administration of [¹⁴C]dolutegravir at a dose of 20 mg (80 μCi). Dolutegravir was well tolerated, and absorption of dolutegravir from the suspension formulation was rapid (median time to peak concentration, 0.5 h), declining in a biphasic fashion. Dolutegravir and the radioactivity had similar terminal plasma half-lives (t_1/2) (15.6 versus 15.7 h), indicating metabolism was formation rate limited with no long-lived metabolites. Only minimal association with blood cellular components was noted with systemic radioactivity. Recovery was essentially complete (mean, 95.6%), with 64.0% and 31.6% of the dose recovered in feces and urine, respectively. Unchanged dolutegravir was the predominant circulating radioactive component in plasma and was consistent with minimal presystemic clearance. Dolutegravir was extensively metabolized. An inactive ether glucuronide, formed primarily via UGT1A1, was the principal biotransformation product at 18.9% of the dose excreted in urine and the principal metabolite in plasma. Two minor biotransformation pathways were oxidation by CYP3A4 (7.9% of the dose) and an oxidative defluorination and glutathione substitution (1.8% of the dose). No disproportionate human metabolites were observed.     

Biochemical Analysis of the Role of G118R-Linked Dolutegravir Drug Resistance Substitutions in HIV-1 Integrase

Drug resistance mutations (DRMs) have been reported for all currently approved anti-HIV drugs, including the latest integrase strand transfer inhibitors (INSTIs). We previously used the new INSTI dolutegravir (DTG) to select a G118R integrase resistance substitution in tissue culture and also showed that secondary substitutions emerged at positions H51Y and E138K. Now, we have characterized the impact of the G118R substitution, alone or in combination with either H51Y or E138K, on 3′ processing and integrase strand transfer activity. The results show that G118R primarily impacted the strand transfer step of integration by diminishing the ability of integrase-long terminal repeat (LTR) complexes to bind target DNA. The addition of H51Y and E138K to G118R partially restored strand transfer activity by modulating the formation of integrase-LTR complexes through increasing LTR DNA affinity and total DNA binding, respectively. This unique mechanism, in which one function of HIV integrase partially compensates for the defect in another function, has not been previously reported. The G118R substitution resulted in low-level resistance to DTG, raltegravir (RAL), and elvitegravir (EVG). The addition of either of H51Y or E138K to G118R did not enhance resistance to DTG, RAL, or EVG. Homology modeling provided insight into the mechanism of resistance conferred by G118R as well as the effects of H51Y or E138K on enzyme activity. The G118R substitution therefore represents a potential avenue for resistance to DTG, similar to that previously described for the R263K substitution. For both pathways, secondary substitutions can lead to either diminished integrase activity and/or increased INSTI susceptibility.     

The R262K Substitution Combined with H51Y in HIV-1 Subtype B Integrase Confers Low-Level Resistance against Dolutegravir

Clinical studies have shown that integrase strand transfer inhibitors (INSTIs) can be used effectively against HIV-1 infection. To date, no resistance substitution has been found in INSTI-naive patients treated with the new integrase inhibitor dolutegravir (DTG). In a recent selection study with DTG, using a virus bearing the H51Y substitution in integrase, the emergence of an R to K substitution at position 262 (R262K) was observed. We characterized this double mutant with respect to integrase strand transfer activity and susceptibility to DTG both biochemically and in tissue culture. We showed that the addition of R262K to H51Y decreased recombinant integrase strand transfer activity but improved integrase DNA-binding affinity, compared to wild-type or H51Y-containing enzymes. The defect in strand transfer activity did not translate into a decrease in HIV-1 infectivity. The combination of H51Y and R262K substitutions slightly decreased susceptibility to DTG (fold change = 1.87) in cell-based resistance assays. Although viral replication was not affected and enzyme efficiency was impaired by the addition of R262K to H51Y, there was an overall increase in the level of biochemical drug resistance against DTG. Our findings suggest that the R at position 262 plays an important role in DNA binding.     

核苷(酸)类似物治疗慢性乙型肝炎患者HBV逆转录酶区变异的研究

目的分析核苷(酸)类似物治疗的慢性乙型肝炎患者HBV逆转录酶区耐药相关变异位点、变异位点组合模式、HBV DNA水平和HBeAg等特征,为临床应用核苷(酸)类似物抗乙肝病毒治疗提供参考。方法纳入研究的141例慢性乙型肝炎患者,使用NAs治疗的107例为治疗组,未使用药物抗病毒治疗的34例为对照组。采用线性探针反向杂交法(PCRRLP)检测HBV逆转录酶区耐药相关变异位点,实时荧光定量PCR法检测乙型肝炎病毒DNA水平,酶联免疫吸附试验检测乙肝免疫标记物,并对耐药相关变异的特点和研究病例的临床资料进行统计学分析。结果核苷(酸)类似物治疗效果不佳组耐药相关变异检出率(82.5%)明显高于未治疗的对照组(5.9%)和抗病毒治疗有效组(有效组10例未检出耐药相关变异)(P0.01)。97例核苷(酸)类似物抗病毒效果不佳的病例中80例检出140个耐药相关变异位点,包括rtM204I 45个、rtM204V 32个、rtA181V 28个、rtL180M 25个、rtN236T 10个;变异位点组合模式主要有:rtL180M+rtM204I、rtM204V、rtM204I+rtM204V、rtA181V+rtM204V、rtM204I、rtA181V+rtN236T等;51个对NAs应答不佳病例耐药变异检出率70.59%,46个病毒学突破病例耐药变异检出率95.65%,差异明显(P0.01)。NAs单药治疗病例早于多药治疗病例检出耐药相关位点(P0.01);NAs治疗时间越长,耐药变异检出率越高(P0.01);病例HBV-DNA水平等级越高,耐药变异检出率也越高(P0.01)。结论⑴NAs治疗慢性乙型肝炎可导致HBV逆转录酶区变异,NAs抗乙型肝炎病毒效果不佳与HBV逆转录酶区变异密切相关;⑵不同NAs药物导致慢性乙肝患者HBV逆转录酶区变异位点、变异模式特点不同;⑶NAs治疗慢性乙型肝炎的用药种类与方式、治疗时长,患者HBV-DNA水平等对HBV逆转录酶区发生耐药变异均可能产生重要影响。提示在NAs治疗前、治疗过程合适时间点检测NAs耐药相关变异位点和HBV DNA水平,可为临床慢性乙型肝炎的抗病毒治疗提供重要参考。