Discussion

Preliminary analysis of an ongoing clinical birth surveillance study suggested increased risk of NTDs in newborns from mothers taking dolutegravir at the time of conception (Zash et al., 2018). Dolutegravir is an integrase inhibitor for the treatment of HIV, a drug class that also includes cabotegravir, bictegravir, raltegravir, and elvitegravir (Han et al., 2017). Folate deficiency increases the incidence rate of NTDs (Daly et al., 1995; Botto et al., 1999; Crider et al., 2014; Kancherla et al., 2018). Folate relies on transport via PCFT for intestinal absorption, FRα-mediated endocytosis along with transport by PCFT and RFC for tissue distribution, including to the fetus, and renal sparing via active tubular reabsorption after extensive glomerular filtration (Fig. 1, Solanky et al., 2010; Zhao et al., 2011).

The aim of the present work was to determine whether dolutegravir and other integrase inhibitor drugs may be clinically relevant inhibitors of the three major folate transport pathways, which would support an increased likelihood of folate deficiency leading to increased NTD risk. Of the five HIV integrase inhibitor drugs evaluated for inhibition of PCFT, RFC, and FRα in folate absorption, distribution, and renal tubular reabsorption, only raltegravir at the highest (i.e., 1.2 g) clinical dose was flagged as a potential clinical inhibitor of PCFT intestinal folate absorption. Otherwise, these five HIV integrase inhibitor drugs are not predicted to inhibit the three major folate transport pathways at clinical therapeutic doses and concentrations (Fig. 6; FDA, 2017).

Based on the current results, dolutegravir is not predicted to elicit clinical decreases in maternal and fetal folate levels. Furthermore, dolutegravir is not an inhibitor of DHFR, the first step in folic acid metabolism in the synthesis of nucleic and amino acids (Cabrera et al., 2019). Taken together, these results do not support dolutegravir-induced folate deficiency as a mechanistic explanation for the reported preliminary observations of increased NTD risk (Zash et al., 2018). As such, these studies do not lend mechanistic support for folic acid supplementation to overcome this proposed increased NTD risk (Zash et al., 2018); however, there is no harm to folic acid supplementation, and it is an established good practice known to reduce the risk of NTDs in the general population who may not otherwise consume adequate amounts of dietary folic acid (Botto et al., 1999; Kancherla et al., 2018). Although studies have repeatedly failed to demonstrate folic acid supplementation to reduce the 4-fold increased NTD rate in epileptic women on valproic acid treatment, folate supplementation is still recommended for women of childbearing potential taking valproic acid as a safe precaution supported by evidence in the general population ((Depakene Prescribing Information, 2011; Hansen et al., 1995; Craig et al., 1999; Candito et al., 2007; 2011)).

HIV integrase inhibitor drug class effect on folate transport pathways was not observed in the present study (Fig. 6), which is conceptually consistent with the lack of clinical evidence for increased NTD risk by this class of drugs ((Viteka Prescribing Information, 2015, Isentress (raltegravir), 2019, Stribild Prescribing Information (2019))). Even raltegravir, which was flagged as a potentially clinically relevant intestinal PCFT inhibitor at the highest 1.2 g clinical dose is not known to increase the clinical risk of NTDs or cause birth defects in preclinical embryofetal toxicology studies (Isentress (raltegravir), 2019).

After the report of potentially increased prevalence of NTDs in the ongoing dolutegravir birth surveillance study (Zash et al., 2018), in vitro FRα receptor binding experiments were used to demonstrate that dolutegravir is a noncompetitive FRα antagonist (Cabrera et al., 2019). Dolutegravir inhibited folic acid binding to FRα up to 44% at concentrations between 16 and 64 μM, with an apparent IC₅₀ of 4.4 μM, where IC₅₀ was defined as dolutegravir concentration eliciting 22% inhibition of folic acid binding to FRα (i.e., half the maximal inhibitory effect) (Cabrera et al., 2019). Considering that Cabrera et al. examined receptor binding as opposed to functional receptor-mediated endocytosis reported in this study, the dolutegravir FRα antagonism results (44% inhibition at dolutegravir concentrations 16–64 μM) are in good agreement with 36% inhibition of FRα-mediated folic acid at 37 μM dolutegravir (Fig. 5D; Fig. 6); however, Cabrera et al. (2019) concluded that dolutegravir’s apparent FRα inhibitory potency of 4.4 μM occurred at therapeutic concentrations based on direct comparison with clinical total plasma concentrations (Table 1; Tivicay, Prescribing Information, 2018). Dolutegravir is highly plasma protein bound (≥98.9%; Table 1; Tivicay, Prescribing Information, 2018), and because only unbound drug is available for pharmacologic activity (i.e., on- and off-target binding, interactions with transporters and drug-metabolizing enzymes, tissue distribution, etc.), direct comparison of in vitro potencies to total plasma concentrations is not clinically pertinent (Smith et al., 2010). Such a direct in vitro to total plasma comparison may have been possible if 4% albumin were added to the buffer in the FRα-binding study to mimic plasma protein binding, but the comparison was made directly between in vitro potency in a protein-free buffer and clinical total plasma concentrations for a highly bound drug (Cabrera et al., 2019), where clinical unbound plasma concentrations are two orders of magnitude lower than total concentrations (Table 1; Tivicay, Prescribing Information, 2018).

Using the accepted clinical extrapolation approach for the dolutegravir apparent FRα inhibitory potency of 4.4 μM in the receptor binding assay, 1 + C_max,u/IC₅₀ = 1.017 (Table 1; FDA, 2017; Cabrera et al., 2019), which is not flagged as clinically relevant at the level of tissue distribution (1 + C_max,u/IC < 1.1) and renal sparing on apical membrane of renal proximal tubules (1 + C_max,u/IC < 1.02) (FDA, 2017). Since 50% FRα antagonism was not achieved, the apparent IC₅₀ is really an IC₂₂ (Cabrera et al., 2019), and it is not accepted practice to use this apparent IC₅₀ parameter in clinical extrapolations (FDA, 2017). Applying the present extrapolation approach to inhibition of folate transport pathways, where the greatest inhibitory potency ≥25% is used wherever 50% inhibition was not achieved, FRα antagonism IC₄₄ = 16 μM (Cabrera et al., 2019), such that 1 + C_max,u/IC₅₀ = 1.005 and well below thresholds for potential clinical significance (Table 1; FDA, 2017; Cabrera et al., 2019).

In the present study, clinical extrapolation of observed in vitro inhibitory potency values were based on established thresholds for clinically relevant transporter inhibition in the intestine (I_gut/IC₅₀ ≥ 10), systemically (1 + C_max,u/IC₅₀ ≥ 1.1), and on the apical membrane of renal proximal tubules (1 + C_max,u/IC₅₀ ≥ 1.02) (FDA, 2017). Although it was validated for transporter-based drug-drug interactions, the principles should apply to drug-nutrient interactions. This extrapolation framework is considered the best available and a conservative approach. As theoretically expected, intensive study of clinical translation of in vitro transport pathway inhibition demonstrated that the same translational pharmacokinetic principles apply to different transporter processes located at the same site in the body (i.e., the same principles and thresholds apply to different transporters localized in the intestine, liver, and basolateral or apical membrane of renal proximal tubules) (Giacomini et al., 2010), Hillgren et al., 2013; FDA, 2017; Zamek-Gliszczynski et al., 2018). Furthermore, these translational approaches have precedent in the extrapolation of in vitro intestinal and systemic thiamine transport by drugs as an alert to drug-induced Wernicke's disease (Giacomini et al., 2017). Notably, these approaches are based on IC₅₀ values where >50% transport function impairment was observed in vitro, and in the present study any inhibitor concentration associated with the greatest extent of inhibition observed ≥25% was reported and extrapolated (Fig. 6). This ≥25% inhibition level was selected on the basis that ≥25% of the cumulative inhibition of folate absorption, distribution, and renal tubular reabsorption (sparing) would decrease fetal folate levels >2-fold (Zamek-Gliszczynski et al., 2009, 2013). The present approach of extrapolating any noted in vitro inhibition ≥25% of folate transport pathways was deliberately conservative and intended to flag any potential clinical inhibition by the five integrase inhibitor drugs. Even with this conservative ≥25% inhibition approach, the present studies do not support 1) dolutegravir as a clinical inhibitor of the three major folate transport pathways or 2) clinically relevant effect of the integrase inhibitor drug class on folate transport.

The present studies are limited to acute inhibition effects by parent drugs. Parent dolutegravir is the predominant circulating moiety with negligible systemic metabolite exposure (Moss et al., 2015). As such, total radioactivity can be assumed to approximate parent drug concentrations, and relevant tissue-to-blood ratios can be estimated from whole-body autoradiography studies in pregnant rats (unpublished data GlaxoSmithKline study 2012N137348_00): placenta-to-blood ratio of 0.59 ± 0.10, fetal-to-maternal blood ratio of 0.13 ± 0.02, and kidney-to-blood ratio of 0.42 ± 0.03. The clinical translational framework based on unbound systemic drug levels is supported by these tissue-to-blood ratios, which do not indicate preferential fetal or renal drug partitioning, as well as systemic exposure primarily to parent dolutegravir and not metabolites; however, potential effects of dolutegravir-glucuronide, the major form of dolutegravir in urine (Moss et al., 2015), on folate tubular reabsorption have not been ruled out by the present studies. Furthermore, potential regulation effects on folate transport and metabolism that may occur upon chronic dolutegravir administration remain to be investigated. These would be most relevant to study clinically in terms of blood folate, folate renal clearance, and maternal-to-neonate blood ratio, because clinical translation of in vitro folate transport and metabolism regulation is not established.