Clinical Trial Report For reprint orders, please contact: [email protected]
Evaluation of the effect of UGT1A1 polymorphisms on dolutegravir pharmacokinetics Aim: To evaluate potential pharmacogenetic effects of UGT1A1 polymorphisms on the pharmacokinetics (PK) of dolutegravir (Tivicay®; ViiV Healthcare, NC, USA), an HIV-1 integrase inhibitor. Patients & methods: Analysis of pooled data from nine Phase I and II clinical studies was undertaken for 89 subjects receiving repeat dolutegravir 50 mg once daily (tablet formulation) who were genotyped for known UGT1A1 functional variants. Results: Geometric mean ratio (92% CI) for subjects carrying low (*28/*28 and *28/*37) and reduced activity (*1/*6, *1/*28, *1/*37, *28/*36 and *36/*37) polymorphisms compared with subjects with normal activity (*1/*1 and *1/*36) showed decreased oral clearance (CL/F; 0.765 [92% CI: 0.659–0.889]), increased area under the concentration–time curve (AUC0-t ; 1.307 [1.125–1.518]) and Cmax (1.221 [1.063–1.402]), respectively. Conclusion: Increased dolutegravir exposure in carriers of UGT1A1 reduced function polymorphisms is not clinically significant based on accumulated safety data so dose adjustment in these individuals is not required. KEYWORDS: dolutegravir n HIV-1 n integrase inhibitor n pharmacogenetic n pharmacokinetic n UGT1A1
Dolutegravir (Tivicay ®; ViiV Healthcare, NC, USA) is an HIV-1 integrase inhibitor (INI) recently approved by the US FDA for the treatment of HIV infection. Its time invariant, 14-h plasma half-life supports once daily (QD) dosing in INI-naive subjects without the need for pharmacokinetic (PK) boosting [1]. Dolutegravir has demonstrated efficacy and safety in treatment-naive subjects with QD dosing [2], and it has also exhibited antiviral activity in subjects who are resistant to INIs [3]. Dolutegravir has no significant CYP450 enzyme inhibition or induction and thus has low drug–drug interaction liabilities [1,4]. It is an inhibitor of OCT2 and thus may increase concentrations of drugs that use this pathway for excretion [4]. Its PK profile is also characterized by low-to-moderate variability and a predictable PK/pharmacodynamic relationship [5]. Dolutegravir is primarily metabolized via UGT1A1 with CYP3A4 contributing to only 10–15% of its metabolism [6]. UGT1A1 is responsible for the glucuronidation of a number of compounds, and functional polymorphisms in the UGT1A1 gene that result in decreased enzyme activity (e.g., *28 and *37 alleles) [7,8] have been shown to affect exposure to irinotecan, raltegravir and raloxifene [9–11]. Such polymorphisms may contribute to safety and dosing recommendations [9]. It is therefore possible that dolutegravir exposure may be affected by functional polymorphisms in UGT1A1.
UGT1A1 is also involved in the conjugation of bilirubin, and genetic predisposition to Gilbert’s syndrome, a condition that can lead to accumulation of unconjugated bilirubin in the plasma, is associated with copy number of a dinucleotide TA(n) repeat polymorphism (rs8175317) in the UGT1A1 promoter. Increasing number of TA repeats is inversely associated with UGT1A1 transcription, with five and six repeats (alleles *36 and *1, respectively) being associated with increased and normal UGT1A1 activity, and seven and eight repeats (alleles *28 and *37, respectively) with low UGT1A1 activity [12,13]. Gilbert’s syndrome is observed in 1–19% of the population [14], and the UGT1A1*28 allele is more common in people of African heritage than those of European heritage and is uncommon in people of Asian heritage [7,15]. This pharmacogenetic (PGx) study has been undertaken to evaluate the effect of UGT1A1 polymorphisms on dolutegravir PK through analysis of pooled subject data from nine clinical studies with standardized dosing and drug formulation.
10.2217/PGS.13.190 © ViiV Healthcare
Pharmacogenomics (2014) 15(1), 9–16
Shuguang Chen1, Pamela St Jean1, Julie Borland1, Ivy Song1, Astrid J Yeo2, Stephen Piscitelli1 & Justin P Rubio*2 GlaxoSmithKline, Research Triangle Park, NC, USA 2 GlaxoSmithKline, Stevenage, Hertfordshire, UK *Author for correspondence: Tel.: +44 0 1438 766 622 Fax: +44 0 1438 762 798 [email protected] 1
Patients & methods Clinical study & subject selection Dolutegravir Phase I (ING111603, ING111604, ING112934, ING113068, ING113096, ING114005, ING114819, ING113099) and Phase IIa (ING111521) clinical studies were selected for inclusion in the analysis because
part of
ISSN 1462-2416
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they were clinical pharmacology repeat-dose studies with intensive PK assessments, used a 50‑mg dose of dolutegravir QD, and the dose was administered as a tablet formulation. Other studies using suspension formulations and those with doses above or below 50 mg QD were not included because it was considered that these additional variables (formulation effects, nonlinear PK) could confound the relationship between dolutegravir PK and potential pharmacogenetic effects. All clinical studies are registered [101]. The studies selected for inclusion were conducted in accordance with good clinical practice procedures, all applicable regulatory requirements and the guiding principles of the Declaration of Helsinki. The study protocols for each clinical study were reviewed and approved by the applicable institutional review board for each clinical site. All subjects provided written informed consent before study entry for the main clinical study. PGx samples were collected under a separate informed consent, or consent section, and this was optional for participants in each study. Blood samples for PGx testing were only collected from those subjects that consented to PGx research. There was no institutional review board review of this analysis since no subjects were enrolled specifically for this study. It was recognized at the commencement of this work that some subjects may have participated in more than one clinical trial. In order to identify these individuals, we employed a genotype-sharing approach using data generated for this project on the Affymetrix® DMETPlus array (CA, USA). For subjects involved in more than one study, the corresponding data were considered as repeated measurements and included in models for analysis. DNA sample collection Venous blood was collected from subjects consenting to PGx research into an EDTA vacutainer. Genomic DNA was extracted using the Gentra Autopure LS automated DNA purification process by one of two central laboratories: Quest Diagnostics (CA, USA) or Quest Diagnostics (Heston, UK). Concentration and quality of the isolated DNA were verified via spectrophotometry and agarose gel electrophoresis by the vendors and GlaxoSmithKline laboratories, respectively. UGT1A1 promoter analysis Genotyping of the UGT1A1 promoter dinucleotide (TA n) repeat polymorphism was performed 10
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by LabCorp (NC, USA) in a Clinical Laboratory Improvement Amendments (CLIA)certified laboratory. PCR with f luorescent labeled primers was used to amplify a genomic segment encompassing the promoter polymorphism from each DNA sample. PCR products were analyzed by capillary electrophoresis on an ABI 3130xl sequencer (Applied Biosystems, Life Technologies Ltd, Paisley, UK) in order to identify which of the four promoter variants were present: UGT1A1 (TA)6 (*1), UGT1A1 (TA)7 (*28), UGT1A1 (TA)5 (*36 ) and UGT1A1 (TA)8 (*37 ). Control DNA samples known to carry specific UGT1A1 genotypes were included to control for the detection of all four TA repeat alleles. Other genotyping Genotyping of UGT1A1*6 (rs4148323) and putative functional variants in CYP3A4, CYP3A5 and PXR was performed by Gen-Probe (Manchester, UK) under research conditions. The Affymetrix DMET-Plus array was used to generate the data for each DNA sample according to manufacturer’s instructions. These data were also used to identify subjects who participated in more than one clinical study (see above). Hardy–Weinberg equilibrium analysis In order to determine assay performance, genotypes for each marker were evaluated for deviation from Hardy–Weinberg equilibrium. Analysis was conducted for each marker separately in the two largest racial subgroups: subjects of European–American or African–American heritage. Robust exact tests (Fisher exact test for biallelic markers and exact tests of proportions for multiallelic markers) were used as genotype frequencies were low in some instances. PK & PGx assessments In each study, dolutegravir PK assessments were performed under fasting conditions or with a moderate fat meal. Steady state PK parameters, area under the concentration–time curve (AUC 0-t), C max and oral clearance (CL/F), obtained from the treatment period for dolutegravir alone (without interacting drug) were evaluated for each study, and were estimated on the basis of intensive serial sampling using noncompartmental methods using WinNonlin Professional Edition v5.2 or above (Pharsight, CA, USA). For each subject, UGT1A1 genotypes were used to predict enzyme activity. future science group
Effect of UGT1A1 polymorphisms on dolutegravir pharmacokinetics
Covariates
Age, sex, study and fasting status were assessed for univariate association with CL/F. Those that were significantly associated (p ≤ 0.05) were included in the model used to test for genetic marker association with CL/F. Age, sex, study and fasting status were assessed for univariate association with each of the three end points in turn (CL/F, Cmax or AUC). Those that were significantly associated (p ≤ 0.05) with a given end point were included in the model used to test for genetic marker association with the corresponding end point. Analysis approach
Analysis of covariance was used to evaluate the main effects of genotype and covariates on CL/F, Cmax and AUC. Using SAS 9.1, a linear mixed effect model was employed with covariates and genetic marker treated as fixed effects and subject as a random effect. To adjust for multiple comparisons, a threshold of a = 0.04 was applied for the primary UGT1A1 ana lysis and a = 0.01 was applied for secondary analyses of putative functional markers in CPY3A4, CYP3A5 and PXR. UGT1A1
The effect of UGT1A1 predicted activity on dolutegravir PK was tested by estimating the ratio of the geometric least square means for CL/F, AUC and Cmax, respectively, in subjects with low (*28/*28, *28/*37 or *37/*37 ) and reduced (*1/*6, *1/*28, *1/*37, *28/*36 and *36/*37 ) UGT1A1 activity (µtest) and subjects with normal (*1/*1 and *1/*36 ) UGT1A1 activity (µ ref ) under the following hypothesis: H0: µtest /µref 2.0 for AUC and Cmax, UGT1A1 effect suggested. Ha: µtest /µ ref ≥0.5 for CL/F or ≤2.0 for AUC and Cmax, no evidence to suggest an effect of UGT1A1. The effect of UGT1A1 activity on dolutegravir PK was also tested in an additive model for the two UGT1A1 polymorphisms, with the number of low-activity alleles for the (TA n) repeat polymorphism (*28 and *37) or for rs4148323 (*6 ) treated as a continuous independent variable under the following hypothesis: H0: µ = 0, number of low activity alleles has no effect on PK. Ha: µ ≠ 0, suggests number of low activity alleles has an effect on PK.
Results Study population This study (ING116265) specifically examined pooled PGx and PK data collected from healthy future science group
Clinical Trial Report
subjects in eight Phase I studies, and from HIVinfected subjects in a Phase IIa monotherapy study, in which dolutegravir was dosed as 50 mg QD using a tablet formulation (please refer to the ‘Patients & methods’ section). Of 139 subjects dosed with a dolutegravir 50 mg QD tablet formulation, 104 (75%) provided informed consent for PGx research. Subsequently, three subjects were found to have insufficient DNA and were not genotyped, and another subject was excluded following geno typing data quality control. Among the remaining 100 subjects, a genotype-sharing approach was used to identify two individuals who participated in three studies, and a further four individuals each participating in two studies (please refer to the ‘Patients & methods’ section). Of the remaining 92 unique individuals, three subjects did not have PK data, thus 89 unique individuals with both genetic and PK data available were included in the final PGx analysis population. The overall mean age of the study population was 36.9 years (standard deviation = 12.1), and the majority of subjects were male (n = 78; 88%). Similar numbers of African–American (n = 37; 42%) and European–American (n = 46; 52%) subjects were available for analysis. Of the remaining six subjects, five had Asian heritage, and one subject had no available ancestry data (Supplementary Table 1; see www.futuremedicine. com/doi/suppl/10.2217/pgs.13.190). Genotyping Table 1 shows the UGT1A1 variants evaluated in this study. Other putative functional variants in CYP3A4, CYP3A5 and PXR, which regulates expression of CYP3A4, were also genotyped because this route of metabolism also plays a role, albeit a minor one, in dolutegravir metabolism [6]. However, as we did not discover an association between any of these variants and dolutegravir PK (data not shown), these data will not be referenced or discussed further. Allele and genotype frequencies were summarized for the entire PGx analysis population and for the European–American and African–American subgroups, respectively. Table 2 shows the distribution of UGT1A1 genotypes, including predicted effect on enzyme activity. A more detailed breakdown of UGT1A1 allele and genotype frequencies is available in Supplementary Table 2. UGT1A1*1 was the most common allele in the analysis population (67%) and in European–American (77%) and African–American (51%) subgroups, respectively. The UGT1A1*28 allele frequency was 27% overall and was www.futuremedicine.com
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Table 1. Genetic markers evaluated. Gene
Polymorphism (star allele)
Variant reference Predicted functional effect sequence (rs) ID
UGT1A1
Promoter region TA repeat (*1 [wt] > *28, *36, *37)
rs8175347
*28 (TA)7 and *37 (TA) 8: reduced activity; *36 (TA) 5: increased activity; compared with wild-type, *1 (TA) 6
UGT1A1
211G>A; Gly71Arg (*1 [wt] > *6)
rs4148323
*6: reduced activity
wt: Wild-type.
more common in African–American (34%) than European–American subjects (23%). The *36 and *37 alleles were only observed in African–American subjects. Predicted UGT1A1 activity was used in the final analysis model and was inferred from genotypes at both the dinucleotide (TA(n)) promoter polymorphism (rs8175347) and a nonsynonymous SNP (UGT1A1*6 ), Gly71Arg (211G>A, rs4148323; Tables 1 & 2). No deviation (p A (rs4148323)
Predicted activity
African– American, n = 37 (%)
European– American, n = 46 (%)
Total, n = 89 (%)
*1/*1
*1/*1
Normal
12 (32)
26 (57)
40 (45)
*1/*36
*1/*1
Normal
1 (3)
0 (0)
1 (1)
Normal subtotal
13 (35)
26 (57)
41 (46)
*1/*1
*1/*6
Reduced
0 (0)
0 (0)
3 (3)
*1/*28
*1/*1
Reduced
12 (32)
19 (41)
31 (35)
*1/*37
*1/*1
Reduced
1 (3)
0 (0)
1 (1)
*28/*36
*1/*1
Reduced
4 (11)
0 (0)
4 (4)
*36/*37
*1/*1
Reduced
1 (3)
0 (0)
1 (1)
Reduced subtotal
18 (49)
19 (41)
40 (45)
*28/*28
*1/*1
Low
4 (11)
1 (2)
6 (7)
*28/*37
*1/*1
Low
1 (3)
0 (0)
1 (1)
Low subtotal
5 (14)
1 (2)
7 (8)
Unknown
1 (3)
0 (0)
1 (1)
*36/*36
*1/*1
†
There is contradictory evidence for an effect of the *36 allele on enzyme activity (increased vs normal) in the literature [13]. †
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Effect of UGT1A1 polymorphisms on dolutegravir pharmacokinetics
Clinical Trial Report
Table 3. Association analysis between pharmacokinetic parameters and UGT1A1 predicted enzyme activity. PK parameter
CL/F (l/h) AUC0-t (µg × h/ml) Cmax (μg/ml)
Geometric LS mean Normal activity (n = 41)
Reduced activity (n = 40)
Low activity (n = 7)
1.09
0.936
0.749
45.7 3.45
53.4 3.89
66.8 4.57
Comparison
Geometric mean ratio (92% CI)
Low + reduced vs normal
0.765 (0.659–0.889)
Low vs normal
0.684 (0.543–0.862)
Low + reduced vs normal
1.307 (1.125–1.518)
Low vs normal
1.462 (1.160–1.842)
Low + reduced vs normal
1.221 (1.063–1.402)
Low vs normal
1.323 (1.068–1.638)
The mixed effect model included predicted enzyme activity, sex and study as fixed effects and subject as a random effect. AUC0-t : Area under the concentration–time curve; CI: Confidence interval; CL/F: Oral clearance; LS: Least squares; PK: Pharmacokinetic.
and Cmax were increased 46 and 32%, respectively. The analysis was repeated for the African–American subgroup alone, and while the trend was consistent with the entire sample for an effect of UGT1A1 enzyme activity on the PK parameters evaluated, no statistically significant associations were observed (Supplementary Table 3). An alternate approach was also used to evaluate potential genetic effects on dolutegravir PK parameters where the number of low-activity alleles carried at each marker was coded as 0, 1 or 2. Only the UGT1A1 promoter poly morphism yielded statistically significant results from this analysis, which were consistent with the analysis based on predicted enzyme activity, whereby each low-activity allele was associated with a modest decrease in CL/F (OR: 0.85; 95% CI: 0.758–0.953), increase in AUC (1.176 [95% CI: 1.049–1.318]) and increase in Cmax (1.134 [95% CI: 1.021–1.259]), respectively.
Discussion UGT1A1 is the principal biotransformation pathway for dolutegravir, while oxidation has a minor role and accounts for only approximately 10% of the dose [16]. There are a number of known polymorphisms in UGT1A1 that are associated with reduced metabolic activity. Therefore, the objective of this analysis was to evaluate whether subjects with genotypes associated with poor or reduced metabolic activity have increased exposure to dolutegravir compared with subjects with genotypes associated with normal metabolic activity. Phase I studies are ideal to evaluate the effect of genetic polymorphisms on drug exposure since subjects often receive intensive PK sampling allowing for determination of all PK parameters. Unfortunately, individual Phase I studies are generally too small to provide sufficient representation of the genotypes of interest and, future science group
therefore, do not provide the basis for an adequately powered analysis. Thus, we performed an analysis of pooled data across nine Phase I and Phase IIa studies encompassing 104 subjects to provide an adequate sample size for a reasonably well-powered study. Of these 104 subjects, we identified a small number who had participated in more than one clinical study or who did not have adequate PK or genetic data available, and consequently, the final sample size included 89 unique subjects. The final study population was primarily of European–American (52%) and African–American (42%) descent. The results of the analysis demonstrate that subjects with genotypes conferring UGT1A1 poor metabolizer status (*28/*28, *28/*37 and *37/*37 ) had a decrease in CL/F (~32%) and an increase in AUC 0-t and Cmax of dolutegravir of approximately 46 and 32%, respectively, compared with subjects with normal UGT1A1 function (*1/*1 and *1/*36 ). These data are similar to those seen with another INI, raltegravir, in which subjects with the *28/*28 genotype had a 40% higher AUC than a group of matched controls with *1/*1 genotypes [10], although UGT1A1*28 is not recognized as a pharmacog enomic biomarker for raltegravir by the FDA. In the current study, it was also found that gender was a significant covariate in the analysis, with female subjects demonstrating approximately 30% lower CL/F than male subjects. The increased exposure we observed for dolutegravir is not considered clinically significant. This position is supported by the observation of no dose-limiting toxicities in a dose-ranging Phase IIb study over 48 weeks [17]. Also, a Phase III trial in treatment-naive subjects (SPRING-2) did not identify any relationship between dolutegravir exposure and adverse events [2]. Furthermore, a QT evaluation www.futuremedicine.com
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at an exposure three- to four-times above that observed with the standard clinical dose also did not show any significant effects [2,18]. Some subjects with higher exposures of dolutegravir have been studied. The standard Phase III dose for dolutegravir is 50 mg QD, although approximately 200 HIV-infected subjects with raltegravir resistance have received 50 mg dolutegravir twice daily [3]. In that study, no specific dose-related toxicities were observed between cohorts receiving 50 mg QD versus those receiving 50 mg twice daily despite the higher dolutegravir exposure. High exposures of dolutegravir have also been seen in healthy subjects receiving atazanavir in combination with dolutegravir. Atazanavir is a known inhibitor of UGT1A1 and increases the mean AUC of dolutegravir by approximately 90% alone or by 62% when boosted by ritonavir [19]. The combination of dolutegravir with atazanavir or atazanavir/ritonavir was generally well tolerated, and no dose adjustment for dolutegravir is required. It is noteworthy that an individual receiving concomitant therapy with atazanavir and dolutegravir would likely have higher exposure to dolutegravir than an individual who receives dolutegravir as monotherapy and is a UGT1A1 poor metabolizer. Across all nine clinical studies, 139 subjects received dolutegravir, but only 104 (75%) gave consent for PGx research. Therefore, a theoretical limitation of this analysis may be a potential bias of the final study population caused by nonconsent to PGx testing by subjects who were treated with dolutegravir. Hence, there could be different genetic and/or PK profiles on average between those subjects who consented to PGx, who are involved in this evaluation, and those from the same clinical studies who did not consent to PGx. The frequency of the UGT1A1*1 and *28 alleles are known to vary widely by ancestry across, and even within, continents [13,20]. However, within our African–American and European–American subgroups, the frequencies of the *28 and *1 alleles were similar to previously reported frequencies, which provides confidence that the results of this evaluation can be extrapolated to the wider population of HIV-infected individuals.
Conclusion Through analysis of pooled data from subjects participating in nine clinical studies, we have shown that UGT1A1 functional polymorphisms have a modest effect on dolutegravir exposure but that these effects are not clinically significant on 14
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the basis of accumulated safety data. These data provide support for the clinical recommendation that dolutegravir does not require dose adjustment or additional clinical monitoring in subjects who carry known functional UGT1A1 polymorphisms.
Future perspective PGx may explain differences during drug development between patients in terms of safety, PK or efficacy, and prospective PGx testing postdrug approval can be used to personalize treatment. A key element of PGx during drug development is having access to DNA samples from subjects who have consented to exploratory research; however, PGx in clinical trials is often an optional rather than a mandatory component of clinical study protocols so patients can opt out of PGx but still participate in the clinical trial. There are several potential ramifications of the optional approach that can significantly impact the ability of PGx to inform decision-making: reduced sample collection resulting from nonconsent will lead to a decrease in study power; ascertainment bias can make it difficult to interpret the results of a subsequent PGx study if the population of consenting subjects is not representative of the entire intent-to-treat population, which can also occur if samples are not collected early in clinical studies. With the increasing recognition by industry and regulators that PGx provides value during drug development, it is likely that a higher proportion of clinical trials in the future will include DNA sample collection and consent for PGx research as a requirement for study entry, rather than an optional consideration. Furthermore, advances in whole genome and custom SNP genotyping platforms are now making it more cost effective to genotype clinical study samples prospectively, banking the genetic data for future analysis, rather than conducting specific studies retrospectively as potential concerns arise. Such an approach would also enable more efficient integration of PGx into decision-making in the dynamic environment of drug development, which would ultimately translate into benefit for patients. Acknowledgements The authors wish to acknowledge the following individuals: our Glaxo S mithKline colleagues, M Mosteller and L Drewett for their support of this work.
Financial & competing interests disclosure Funding for these studies was provided by ViiV Healthcare. All listed authors meet the criteria for authorship set forth by the International Committee for Medical Journal Editors. future science group
Clinical Trial Report
Effect of UGT1A1 polymorphisms on dolutegravir pharmacokinetics
S Chen, P St Jean, J Borland, I Song, AJ Yeo, S Piscitelli and JP Rubio are employees of GlaxoSmithKline and own stock in GlaxoSmithKline. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. The authors also wish to acknowledge the following individuals for writing assistance during the development of this manuscript: J Rossi and P Farmer (MedThink
SciCom). Funding for this writing assistance was provided by ViiV Healthcare.
Ethical conduct of research The authors state that they have obtained appropriate insti tutional review board approval or have followed the princi ples outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.
Executive summary Dolutegravir (Tivicay®; ViiV Healthcare, NC, USA) is a recently approved HIV-1 integrase inhibitor. UGT1A1 functional polymorphisms can affect the metabolism of certain drugs in humans, potentially leading to altered drug exposure, safety and efficacy. Dolutegravir is primarily metabolized via UGT1A1 so the objective of this study was to investigate the prospect that UGT1A1 functional polymorphisms may influence dolutegravir exposure. A pooled analysis of nine Phase I and II clinical studies was undertaken for 89 subjects receiving repeat dolutegravir 50 mg once daily (tablet formulation) who were genotyped for known functional variants. The results of this investigation showed that while UGT1A1 functional polymorphisms have a modest effect on dolutegravir exposure, these effects are not clinically significant in light of the accumulated safety data. These data provide support for the clinical recommendation that dolutegravir does not require dose adjustment or additional clinical monitoring in subjects who carry known functional UGT1A1 polymorphisms.
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future science group
Evaluation of the effect of UGT1A1 polymorphisms on dolutegravir pharmacokinetics Aim: To evaluate potential pharmacogenetic effects of UGT1A1 polymorphisms on the pharmacokinetics (PK) of dolutegravir (Tivicay®; ViiV Healthcare, NC, USA), an HIV-1 integrase inhibitor. Patients & methods: Analysis of pooled data from nine Phase I and II clinical studies was undertaken for 89 subjects receiving repeat dolutegravir 50 mg once daily (tablet formulation) who were genotyped for known UGT1A1 functional variants. Results: Geometric mean ratio (92% CI) for subjects carrying low (*28/*28 and *28/*37) and reduced activity (*1/*6, *1/*28, *1/*37, *28/*36 and *36/*37) polymorphisms compared with subjects with normal activity (*1/*1 and *1/*36) showed decreased oral clearance (CL/F; 0.765 [92% CI: 0.659–0.889]), increased area under the concentration–time curve (AUC0-t ; 1.307 [1.125–1.518]) and Cmax (1.221 [1.063–1.402]), respectively. Conclusion: Increased dolutegravir exposure in carriers of UGT1A1 reduced function polymorphisms is not clinically significant based on accumulated safety data so dose adjustment in these individuals is not required. KEYWORDS: dolutegravir n HIV-1 n integrase inhibitor n pharmacogenetic n pharmacokinetic n UGT1A1
Dolutegravir (Tivicay ®; ViiV Healthcare, NC, USA) is an HIV-1 integrase inhibitor (INI) recently approved by the US FDA for the treatment of HIV infection. Its time invariant, 14-h plasma half-life supports once daily (QD) dosing in INI-naive subjects without the need for pharmacokinetic (PK) boosting [1]. Dolutegravir has demonstrated efficacy and safety in treatment-naive subjects with QD dosing [2], and it has also exhibited antiviral activity in subjects who are resistant to INIs [3]. Dolutegravir has no significant CYP450 enzyme inhibition or induction and thus has low drug–drug interaction liabilities [1,4]. It is an inhibitor of OCT2 and thus may increase concentrations of drugs that use this pathway for excretion [4]. Its PK profile is also characterized by low-to-moderate variability and a predictable PK/pharmacodynamic relationship [5]. Dolutegravir is primarily metabolized via UGT1A1 with CYP3A4 contributing to only 10–15% of its metabolism [6]. UGT1A1 is responsible for the glucuronidation of a number of compounds, and functional polymorphisms in the UGT1A1 gene that result in decreased enzyme activity (e.g., *28 and *37 alleles) [7,8] have been shown to affect exposure to irinotecan, raltegravir and raloxifene [9–11]. Such polymorphisms may contribute to safety and dosing recommendations [9]. It is therefore possible that dolutegravir exposure may be affected by functional polymorphisms in UGT1A1.
UGT1A1 is also involved in the conjugation of bilirubin, and genetic predisposition to Gilbert’s syndrome, a condition that can lead to accumulation of unconjugated bilirubin in the plasma, is associated with copy number of a dinucleotide TA(n) repeat polymorphism (rs8175317) in the UGT1A1 promoter. Increasing number of TA repeats is inversely associated with UGT1A1 transcription, with five and six repeats (alleles *36 and *1, respectively) being associated with increased and normal UGT1A1 activity, and seven and eight repeats (alleles *28 and *37, respectively) with low UGT1A1 activity [12,13]. Gilbert’s syndrome is observed in 1–19% of the population [14], and the UGT1A1*28 allele is more common in people of African heritage than those of European heritage and is uncommon in people of Asian heritage [7,15]. This pharmacogenetic (PGx) study has been undertaken to evaluate the effect of UGT1A1 polymorphisms on dolutegravir PK through analysis of pooled subject data from nine clinical studies with standardized dosing and drug formulation.
10.2217/PGS.13.190 © ViiV Healthcare
Pharmacogenomics (2014) 15(1), 9–16
Shuguang Chen1, Pamela St Jean1, Julie Borland1, Ivy Song1, Astrid J Yeo2, Stephen Piscitelli1 & Justin P Rubio*2 GlaxoSmithKline, Research Triangle Park, NC, USA 2 GlaxoSmithKline, Stevenage, Hertfordshire, UK *Author for correspondence: Tel.: +44 0 1438 766 622 Fax: +44 0 1438 762 798 [email protected] 1
Patients & methods Clinical study & subject selection Dolutegravir Phase I (ING111603, ING111604, ING112934, ING113068, ING113096, ING114005, ING114819, ING113099) and Phase IIa (ING111521) clinical studies were selected for inclusion in the analysis because
part of
ISSN 1462-2416
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they were clinical pharmacology repeat-dose studies with intensive PK assessments, used a 50‑mg dose of dolutegravir QD, and the dose was administered as a tablet formulation. Other studies using suspension formulations and those with doses above or below 50 mg QD were not included because it was considered that these additional variables (formulation effects, nonlinear PK) could confound the relationship between dolutegravir PK and potential pharmacogenetic effects. All clinical studies are registered [101]. The studies selected for inclusion were conducted in accordance with good clinical practice procedures, all applicable regulatory requirements and the guiding principles of the Declaration of Helsinki. The study protocols for each clinical study were reviewed and approved by the applicable institutional review board for each clinical site. All subjects provided written informed consent before study entry for the main clinical study. PGx samples were collected under a separate informed consent, or consent section, and this was optional for participants in each study. Blood samples for PGx testing were only collected from those subjects that consented to PGx research. There was no institutional review board review of this analysis since no subjects were enrolled specifically for this study. It was recognized at the commencement of this work that some subjects may have participated in more than one clinical trial. In order to identify these individuals, we employed a genotype-sharing approach using data generated for this project on the Affymetrix® DMETPlus array (CA, USA). For subjects involved in more than one study, the corresponding data were considered as repeated measurements and included in models for analysis. DNA sample collection Venous blood was collected from subjects consenting to PGx research into an EDTA vacutainer. Genomic DNA was extracted using the Gentra Autopure LS automated DNA purification process by one of two central laboratories: Quest Diagnostics (CA, USA) or Quest Diagnostics (Heston, UK). Concentration and quality of the isolated DNA were verified via spectrophotometry and agarose gel electrophoresis by the vendors and GlaxoSmithKline laboratories, respectively. UGT1A1 promoter analysis Genotyping of the UGT1A1 promoter dinucleotide (TA n) repeat polymorphism was performed 10
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by LabCorp (NC, USA) in a Clinical Laboratory Improvement Amendments (CLIA)certified laboratory. PCR with f luorescent labeled primers was used to amplify a genomic segment encompassing the promoter polymorphism from each DNA sample. PCR products were analyzed by capillary electrophoresis on an ABI 3130xl sequencer (Applied Biosystems, Life Technologies Ltd, Paisley, UK) in order to identify which of the four promoter variants were present: UGT1A1 (TA)6 (*1), UGT1A1 (TA)7 (*28), UGT1A1 (TA)5 (*36 ) and UGT1A1 (TA)8 (*37 ). Control DNA samples known to carry specific UGT1A1 genotypes were included to control for the detection of all four TA repeat alleles. Other genotyping Genotyping of UGT1A1*6 (rs4148323) and putative functional variants in CYP3A4, CYP3A5 and PXR was performed by Gen-Probe (Manchester, UK) under research conditions. The Affymetrix DMET-Plus array was used to generate the data for each DNA sample according to manufacturer’s instructions. These data were also used to identify subjects who participated in more than one clinical study (see above). Hardy–Weinberg equilibrium analysis In order to determine assay performance, genotypes for each marker were evaluated for deviation from Hardy–Weinberg equilibrium. Analysis was conducted for each marker separately in the two largest racial subgroups: subjects of European–American or African–American heritage. Robust exact tests (Fisher exact test for biallelic markers and exact tests of proportions for multiallelic markers) were used as genotype frequencies were low in some instances. PK & PGx assessments In each study, dolutegravir PK assessments were performed under fasting conditions or with a moderate fat meal. Steady state PK parameters, area under the concentration–time curve (AUC 0-t), C max and oral clearance (CL/F), obtained from the treatment period for dolutegravir alone (without interacting drug) were evaluated for each study, and were estimated on the basis of intensive serial sampling using noncompartmental methods using WinNonlin Professional Edition v5.2 or above (Pharsight, CA, USA). For each subject, UGT1A1 genotypes were used to predict enzyme activity. future science group
Effect of UGT1A1 polymorphisms on dolutegravir pharmacokinetics
Covariates
Age, sex, study and fasting status were assessed for univariate association with CL/F. Those that were significantly associated (p ≤ 0.05) were included in the model used to test for genetic marker association with CL/F. Age, sex, study and fasting status were assessed for univariate association with each of the three end points in turn (CL/F, Cmax or AUC). Those that were significantly associated (p ≤ 0.05) with a given end point were included in the model used to test for genetic marker association with the corresponding end point. Analysis approach
Analysis of covariance was used to evaluate the main effects of genotype and covariates on CL/F, Cmax and AUC. Using SAS 9.1, a linear mixed effect model was employed with covariates and genetic marker treated as fixed effects and subject as a random effect. To adjust for multiple comparisons, a threshold of a = 0.04 was applied for the primary UGT1A1 ana lysis and a = 0.01 was applied for secondary analyses of putative functional markers in CPY3A4, CYP3A5 and PXR. UGT1A1
The effect of UGT1A1 predicted activity on dolutegravir PK was tested by estimating the ratio of the geometric least square means for CL/F, AUC and Cmax, respectively, in subjects with low (*28/*28, *28/*37 or *37/*37 ) and reduced (*1/*6, *1/*28, *1/*37, *28/*36 and *36/*37 ) UGT1A1 activity (µtest) and subjects with normal (*1/*1 and *1/*36 ) UGT1A1 activity (µ ref ) under the following hypothesis: H0: µtest /µref 2.0 for AUC and Cmax, UGT1A1 effect suggested. Ha: µtest /µ ref ≥0.5 for CL/F or ≤2.0 for AUC and Cmax, no evidence to suggest an effect of UGT1A1. The effect of UGT1A1 activity on dolutegravir PK was also tested in an additive model for the two UGT1A1 polymorphisms, with the number of low-activity alleles for the (TA n) repeat polymorphism (*28 and *37) or for rs4148323 (*6 ) treated as a continuous independent variable under the following hypothesis: H0: µ = 0, number of low activity alleles has no effect on PK. Ha: µ ≠ 0, suggests number of low activity alleles has an effect on PK.
Results Study population This study (ING116265) specifically examined pooled PGx and PK data collected from healthy future science group
Clinical Trial Report
subjects in eight Phase I studies, and from HIVinfected subjects in a Phase IIa monotherapy study, in which dolutegravir was dosed as 50 mg QD using a tablet formulation (please refer to the ‘Patients & methods’ section). Of 139 subjects dosed with a dolutegravir 50 mg QD tablet formulation, 104 (75%) provided informed consent for PGx research. Subsequently, three subjects were found to have insufficient DNA and were not genotyped, and another subject was excluded following geno typing data quality control. Among the remaining 100 subjects, a genotype-sharing approach was used to identify two individuals who participated in three studies, and a further four individuals each participating in two studies (please refer to the ‘Patients & methods’ section). Of the remaining 92 unique individuals, three subjects did not have PK data, thus 89 unique individuals with both genetic and PK data available were included in the final PGx analysis population. The overall mean age of the study population was 36.9 years (standard deviation = 12.1), and the majority of subjects were male (n = 78; 88%). Similar numbers of African–American (n = 37; 42%) and European–American (n = 46; 52%) subjects were available for analysis. Of the remaining six subjects, five had Asian heritage, and one subject had no available ancestry data (Supplementary Table 1; see www.futuremedicine. com/doi/suppl/10.2217/pgs.13.190). Genotyping Table 1 shows the UGT1A1 variants evaluated in this study. Other putative functional variants in CYP3A4, CYP3A5 and PXR, which regulates expression of CYP3A4, were also genotyped because this route of metabolism also plays a role, albeit a minor one, in dolutegravir metabolism [6]. However, as we did not discover an association between any of these variants and dolutegravir PK (data not shown), these data will not be referenced or discussed further. Allele and genotype frequencies were summarized for the entire PGx analysis population and for the European–American and African–American subgroups, respectively. Table 2 shows the distribution of UGT1A1 genotypes, including predicted effect on enzyme activity. A more detailed breakdown of UGT1A1 allele and genotype frequencies is available in Supplementary Table 2. UGT1A1*1 was the most common allele in the analysis population (67%) and in European–American (77%) and African–American (51%) subgroups, respectively. The UGT1A1*28 allele frequency was 27% overall and was www.futuremedicine.com
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Table 1. Genetic markers evaluated. Gene
Polymorphism (star allele)
Variant reference Predicted functional effect sequence (rs) ID
UGT1A1
Promoter region TA repeat (*1 [wt] > *28, *36, *37)
rs8175347
*28 (TA)7 and *37 (TA) 8: reduced activity; *36 (TA) 5: increased activity; compared with wild-type, *1 (TA) 6
UGT1A1
211G>A; Gly71Arg (*1 [wt] > *6)
rs4148323
*6: reduced activity
wt: Wild-type.
more common in African–American (34%) than European–American subjects (23%). The *36 and *37 alleles were only observed in African–American subjects. Predicted UGT1A1 activity was used in the final analysis model and was inferred from genotypes at both the dinucleotide (TA(n)) promoter polymorphism (rs8175347) and a nonsynonymous SNP (UGT1A1*6 ), Gly71Arg (211G>A, rs4148323; Tables 1 & 2). No deviation (p A (rs4148323)
Predicted activity
African– American, n = 37 (%)
European– American, n = 46 (%)
Total, n = 89 (%)
*1/*1
*1/*1
Normal
12 (32)
26 (57)
40 (45)
*1/*36
*1/*1
Normal
1 (3)
0 (0)
1 (1)
Normal subtotal
13 (35)
26 (57)
41 (46)
*1/*1
*1/*6
Reduced
0 (0)
0 (0)
3 (3)
*1/*28
*1/*1
Reduced
12 (32)
19 (41)
31 (35)
*1/*37
*1/*1
Reduced
1 (3)
0 (0)
1 (1)
*28/*36
*1/*1
Reduced
4 (11)
0 (0)
4 (4)
*36/*37
*1/*1
Reduced
1 (3)
0 (0)
1 (1)
Reduced subtotal
18 (49)
19 (41)
40 (45)
*28/*28
*1/*1
Low
4 (11)
1 (2)
6 (7)
*28/*37
*1/*1
Low
1 (3)
0 (0)
1 (1)
Low subtotal
5 (14)
1 (2)
7 (8)
Unknown
1 (3)
0 (0)
1 (1)
*36/*36
*1/*1
†
There is contradictory evidence for an effect of the *36 allele on enzyme activity (increased vs normal) in the literature [13]. †
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Effect of UGT1A1 polymorphisms on dolutegravir pharmacokinetics
Clinical Trial Report
Table 3. Association analysis between pharmacokinetic parameters and UGT1A1 predicted enzyme activity. PK parameter
CL/F (l/h) AUC0-t (µg × h/ml) Cmax (μg/ml)
Geometric LS mean Normal activity (n = 41)
Reduced activity (n = 40)
Low activity (n = 7)
1.09
0.936
0.749
45.7 3.45
53.4 3.89
66.8 4.57
Comparison
Geometric mean ratio (92% CI)
Low + reduced vs normal
0.765 (0.659–0.889)
Low vs normal
0.684 (0.543–0.862)
Low + reduced vs normal
1.307 (1.125–1.518)
Low vs normal
1.462 (1.160–1.842)
Low + reduced vs normal
1.221 (1.063–1.402)
Low vs normal
1.323 (1.068–1.638)
The mixed effect model included predicted enzyme activity, sex and study as fixed effects and subject as a random effect. AUC0-t : Area under the concentration–time curve; CI: Confidence interval; CL/F: Oral clearance; LS: Least squares; PK: Pharmacokinetic.
and Cmax were increased 46 and 32%, respectively. The analysis was repeated for the African–American subgroup alone, and while the trend was consistent with the entire sample for an effect of UGT1A1 enzyme activity on the PK parameters evaluated, no statistically significant associations were observed (Supplementary Table 3). An alternate approach was also used to evaluate potential genetic effects on dolutegravir PK parameters where the number of low-activity alleles carried at each marker was coded as 0, 1 or 2. Only the UGT1A1 promoter poly morphism yielded statistically significant results from this analysis, which were consistent with the analysis based on predicted enzyme activity, whereby each low-activity allele was associated with a modest decrease in CL/F (OR: 0.85; 95% CI: 0.758–0.953), increase in AUC (1.176 [95% CI: 1.049–1.318]) and increase in Cmax (1.134 [95% CI: 1.021–1.259]), respectively.
Discussion UGT1A1 is the principal biotransformation pathway for dolutegravir, while oxidation has a minor role and accounts for only approximately 10% of the dose [16]. There are a number of known polymorphisms in UGT1A1 that are associated with reduced metabolic activity. Therefore, the objective of this analysis was to evaluate whether subjects with genotypes associated with poor or reduced metabolic activity have increased exposure to dolutegravir compared with subjects with genotypes associated with normal metabolic activity. Phase I studies are ideal to evaluate the effect of genetic polymorphisms on drug exposure since subjects often receive intensive PK sampling allowing for determination of all PK parameters. Unfortunately, individual Phase I studies are generally too small to provide sufficient representation of the genotypes of interest and, future science group
therefore, do not provide the basis for an adequately powered analysis. Thus, we performed an analysis of pooled data across nine Phase I and Phase IIa studies encompassing 104 subjects to provide an adequate sample size for a reasonably well-powered study. Of these 104 subjects, we identified a small number who had participated in more than one clinical study or who did not have adequate PK or genetic data available, and consequently, the final sample size included 89 unique subjects. The final study population was primarily of European–American (52%) and African–American (42%) descent. The results of the analysis demonstrate that subjects with genotypes conferring UGT1A1 poor metabolizer status (*28/*28, *28/*37 and *37/*37 ) had a decrease in CL/F (~32%) and an increase in AUC 0-t and Cmax of dolutegravir of approximately 46 and 32%, respectively, compared with subjects with normal UGT1A1 function (*1/*1 and *1/*36 ). These data are similar to those seen with another INI, raltegravir, in which subjects with the *28/*28 genotype had a 40% higher AUC than a group of matched controls with *1/*1 genotypes [10], although UGT1A1*28 is not recognized as a pharmacog enomic biomarker for raltegravir by the FDA. In the current study, it was also found that gender was a significant covariate in the analysis, with female subjects demonstrating approximately 30% lower CL/F than male subjects. The increased exposure we observed for dolutegravir is not considered clinically significant. This position is supported by the observation of no dose-limiting toxicities in a dose-ranging Phase IIb study over 48 weeks [17]. Also, a Phase III trial in treatment-naive subjects (SPRING-2) did not identify any relationship between dolutegravir exposure and adverse events [2]. Furthermore, a QT evaluation www.futuremedicine.com
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at an exposure three- to four-times above that observed with the standard clinical dose also did not show any significant effects [2,18]. Some subjects with higher exposures of dolutegravir have been studied. The standard Phase III dose for dolutegravir is 50 mg QD, although approximately 200 HIV-infected subjects with raltegravir resistance have received 50 mg dolutegravir twice daily [3]. In that study, no specific dose-related toxicities were observed between cohorts receiving 50 mg QD versus those receiving 50 mg twice daily despite the higher dolutegravir exposure. High exposures of dolutegravir have also been seen in healthy subjects receiving atazanavir in combination with dolutegravir. Atazanavir is a known inhibitor of UGT1A1 and increases the mean AUC of dolutegravir by approximately 90% alone or by 62% when boosted by ritonavir [19]. The combination of dolutegravir with atazanavir or atazanavir/ritonavir was generally well tolerated, and no dose adjustment for dolutegravir is required. It is noteworthy that an individual receiving concomitant therapy with atazanavir and dolutegravir would likely have higher exposure to dolutegravir than an individual who receives dolutegravir as monotherapy and is a UGT1A1 poor metabolizer. Across all nine clinical studies, 139 subjects received dolutegravir, but only 104 (75%) gave consent for PGx research. Therefore, a theoretical limitation of this analysis may be a potential bias of the final study population caused by nonconsent to PGx testing by subjects who were treated with dolutegravir. Hence, there could be different genetic and/or PK profiles on average between those subjects who consented to PGx, who are involved in this evaluation, and those from the same clinical studies who did not consent to PGx. The frequency of the UGT1A1*1 and *28 alleles are known to vary widely by ancestry across, and even within, continents [13,20]. However, within our African–American and European–American subgroups, the frequencies of the *28 and *1 alleles were similar to previously reported frequencies, which provides confidence that the results of this evaluation can be extrapolated to the wider population of HIV-infected individuals.
Conclusion Through analysis of pooled data from subjects participating in nine clinical studies, we have shown that UGT1A1 functional polymorphisms have a modest effect on dolutegravir exposure but that these effects are not clinically significant on 14
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the basis of accumulated safety data. These data provide support for the clinical recommendation that dolutegravir does not require dose adjustment or additional clinical monitoring in subjects who carry known functional UGT1A1 polymorphisms.
Future perspective PGx may explain differences during drug development between patients in terms of safety, PK or efficacy, and prospective PGx testing postdrug approval can be used to personalize treatment. A key element of PGx during drug development is having access to DNA samples from subjects who have consented to exploratory research; however, PGx in clinical trials is often an optional rather than a mandatory component of clinical study protocols so patients can opt out of PGx but still participate in the clinical trial. There are several potential ramifications of the optional approach that can significantly impact the ability of PGx to inform decision-making: reduced sample collection resulting from nonconsent will lead to a decrease in study power; ascertainment bias can make it difficult to interpret the results of a subsequent PGx study if the population of consenting subjects is not representative of the entire intent-to-treat population, which can also occur if samples are not collected early in clinical studies. With the increasing recognition by industry and regulators that PGx provides value during drug development, it is likely that a higher proportion of clinical trials in the future will include DNA sample collection and consent for PGx research as a requirement for study entry, rather than an optional consideration. Furthermore, advances in whole genome and custom SNP genotyping platforms are now making it more cost effective to genotype clinical study samples prospectively, banking the genetic data for future analysis, rather than conducting specific studies retrospectively as potential concerns arise. Such an approach would also enable more efficient integration of PGx into decision-making in the dynamic environment of drug development, which would ultimately translate into benefit for patients. Acknowledgements The authors wish to acknowledge the following individuals: our Glaxo S mithKline colleagues, M Mosteller and L Drewett for their support of this work.
Financial & competing interests disclosure Funding for these studies was provided by ViiV Healthcare. All listed authors meet the criteria for authorship set forth by the International Committee for Medical Journal Editors. future science group
Clinical Trial Report
Effect of UGT1A1 polymorphisms on dolutegravir pharmacokinetics
S Chen, P St Jean, J Borland, I Song, AJ Yeo, S Piscitelli and JP Rubio are employees of GlaxoSmithKline and own stock in GlaxoSmithKline. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. The authors also wish to acknowledge the following individuals for writing assistance during the development of this manuscript: J Rossi and P Farmer (MedThink
SciCom). Funding for this writing assistance was provided by ViiV Healthcare.
Ethical conduct of research The authors state that they have obtained appropriate insti tutional review board approval or have followed the princi ples outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.
Executive summary Dolutegravir (Tivicay®; ViiV Healthcare, NC, USA) is a recently approved HIV-1 integrase inhibitor. UGT1A1 functional polymorphisms can affect the metabolism of certain drugs in humans, potentially leading to altered drug exposure, safety and efficacy. Dolutegravir is primarily metabolized via UGT1A1 so the objective of this study was to investigate the prospect that UGT1A1 functional polymorphisms may influence dolutegravir exposure. A pooled analysis of nine Phase I and II clinical studies was undertaken for 89 subjects receiving repeat dolutegravir 50 mg once daily (tablet formulation) who were genotyped for known functional variants. The results of this investigation showed that while UGT1A1 functional polymorphisms have a modest effect on dolutegravir exposure, these effects are not clinically significant in light of the accumulated safety data. These data provide support for the clinical recommendation that dolutegravir does not require dose adjustment or additional clinical monitoring in subjects who carry known functional UGT1A1 polymorphisms.
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