Impact of UGT1A1 gene polymorphisms on plasma dolutegravir trough concentrations and neuropsychiatric adverse events in Japanese individuals infected with HIV-1

This study included Japanese patients infected with HIV-1 who visited the National Hospital Organization Osaka National Hospital, who were 20 years old or older, and who received anti-HIV therapy in combination with DTG. Written consent was acquired prior to sample collection and testing. The subjects were limited to those taking DTG in the morning, because of the time limitations of our institution on the collection and processing of patient samples. We interviewed them about their medication adherence to exclude subjects with poor adherence. Subjects concomitantly using a potent inhibitor of UGT1A1 (atazanavir), potent inducers of UGT1A1, CYP3A, or both (rifampicin, carbamazepine, phenytoin, and phenobarbital), potent inhibitors of CYP3A (itraconazole, cobicistat, and ritonavir), and any other drugs that could affect plasma DTG concentrations (aluminum- or magnesium-containing antacids or both) were excluded.

Saliva samples were collected from the patients, absorbed with a filter paper, and subsequently dried. A portion of the filter paper containing the saliva sample was cut and used as a template in the polymerase chain reaction (PCR) used to determine the presence or absence of gene polymorphisms using a previously reported method [21].

Blood samples were collected after oral administration of DTG for 10 days or longer. Whole blood samples (5 mL) were collected in test tubes containing heparin sodium 22–26 h after an oral administration of DTG. The blood samples were centrifuged at 1200×g for 10 min to isolate 2 mL plasma, which was stored in a freezer at −80°C until assayed. We measured the DTG plasma concentration using mass spectrometry based on our previously developed assay for raltegravir (RAL) [22], an integrase inhibitor. DTG was detected at a mass-to-charge ratio (m/z) of 420 using the MassLynx analysis software version 4.0 (Waters Corp., Milford, MA).

Five symptoms (dizziness, headache, insomnia, restlessness, and anxiety) were selected as NP-AEs. The information on NP-AEs was obtained upon patient entry into this study, and the subjects were followed-up until March 2017 or the discontinuation of DTG. NP-AEs, which had developed after starting DTG but had ceased by initiation of the study, were included in the analyses. Plasma DTG trough concentrations were compared between the subjects with or without NP-AEs.

For group comparisons, the Wilcoxon rank-sum test was used. If a statistically significant result was obtained for multiple group comparison, Steel’s test was used as the post-hoc test. To search for factors associated with trough concentrations of DTG, multivariate analysis using logistic regression was conducted. The subjects were classified into two groups using the median trough DTG concentration (1.06 μg/mL) as a cut-off value. The variables used were age (over 40 years old), weight (less than 60 kg), CD4 count (less than 500/μL), and the number of *6 and *28 alleles; they were analyzed using the forced input method. The cut-off value for age was set to 40 years old to match that of a previously published study [8]. To search for factors associated with selected NP-AEs, the cumulative incidence of NP-AEs was estimated by the Kaplan-Meier method. The subjects were divided into two groups, stratified by the presence or absence of carrying UGT1A1*6, UGT1A1*28, or both alleles (normal allele group, −/− versus reduced-function allele group, −/*28, −/*6, *28/*28, *6/28, and *6/*6), and these two groups were compared. We adopted a significance level of 5%, and the statistical analysis was conducted using JMP Software version 10.0.0 (SAS Institute Inc., Cary, NC). The p value of χ² in the analyses of Hardy-Weinberg equilibrium was calculated using R software, version 3.3.3 (R Foundation for Statistical Computing, Vienna, Austria).

Table 1 shows the general characteristics of the 107 individuals infected with HIV-1 who participated in this study based on the type of UGT1A1 gene polymorphism. The subjects comprised eight (7%) *6 homozygotes, three (3%) *28 homozygotes, four (4%) *6/*28 compound heterozygotes, 23 (21%) *6 heterozygotes, 18 (17%) *28 heterozygotes, and 51 (48%) patients homozygous for the wildtype UGT1A1 genotype. The *6 and *28 allele frequencies were 20% and 13%, respectively. Distribution of the *6 and *28 allele was in the Hardy-Weinberg equilibrium (p = 0.83 and p = 0.92, respectively). Table 1 shows that there were no significant differences among UGT1A1 gene polymorphism frequencies with respect to the listed participant characteristics, except for rilpivirine use that was due to one individual.

The median DTG trough concentration of the 107 subjects was 1.06 μg/mL. First, the DTG trough concentrations were classified based on UGT1A1 gene polymorphisms and group comparisons were conducted (Fig. 1a, Wilcoxon rank-sum test, p = 0.0008). The median DTG trough concentration of the *6 homozygous patients (1.43 μg/mL) was significantly higher than that of patients carrying both normal alleles (0.82 μg/mL, p = 0.0054). The median concentrations of the *6 and *28 heterozygous patients (1.29 and 1.2 μg/mL, respectively) were also significantly higher than that of the homozygous patients carrying normal alleles (p = 0.0223 and p = 0.0254, respectively). Moreover, no statistically significant difference was detected in median DTG trough concentrations between patients with both normal alleles and those with both *28 alleles (1.18 μg/mL) or between those with both normal alleles and *6/*28 compound heterozygotes (1.37 μg/mL). However, we could not rule out the possibility that the statistical power was insufficient due to the small numbers of patients.

Next, a multivariate logistic regression analysis was conducted using the median DTG trough concentration, 1.06 μg/mL, as the cut-off value to find factors associated with high DTG trough concentrations. Carrying one or two UGT1A1*6 gene alleles and a UGT1A1*28 allele, as well as an age < 40 years were found to be independent factors associated with high DTG trough concentrations (Table 2).

During the follow-up period (255 person-years), selected NP-AEs after the use of DTG were observed in 34 subjects (32%, Table 3). Insomnia was the most frequent (n = 14, 13%) among selected NP-AEs, followed by dizziness (n = 9, 8%), and headache (n = 9, 8%). The severity of all NP-AEs was grade 1 or 2. Six subjects discontinued DTG, and all were because of NP-AEs. Two subjects were lost to follow-up because of personal reasons. As shown in Fig. 1b, plasma DTG trough concentration was significantly higher in the patients with NP-AEs (median, 1.31 μg/mL) than in those without NP-AEs (median, 1.01 μg/mL, Wilcoxon rank-sum test, p = 0.0013). Next, we estimated the factors associated with the cumulative incidence of selected NP-AEs by use of Kaplan-Meier curves (Fig. 2). Plasma DTG trough concentration (≥ 1.06 μg/mL), and carrying UGT1A1*6, UGT1A1*28, or both alleles were associated with higher cumulative incidences of selected NP-AEs (log rank-test, p = 0.0105, and 0.0454, respectively). Age did not show this association (log rank-test, p = 0.2477).