Long-acting dolutegravir formulations prevent neurodevelopmental impairments in a mouse model

Abstract

The World Health Organization has recommended dolutegravir (DTG) as a preferred first-line treatment for treatment naive and experienced people living with human immunodeficiency virus type one (PLWHIV). Based on these recommendations 15 million PLWHIV worldwide are expected to be treated with DTG regimens on or before 2025. This includes pregnant women. Current widespread use of DTG is linked to the drug's high potency, barrier to resistance, and cost-effectiveness. Despite such benefits, potential risks of DTG-linked fetal neurodevelopmental toxicity remain a concern. To this end, novel formulation strategies are urgently needed in order to maximize DTG's therapeutic potentials while limiting adverse events. In regard to potential maternal fetal toxicities, we hypothesized that injectable long-acting nanoformulated DTG (NDTG) could provide improved safety by reducing drug fetal exposures compared to orally administered native drug. To test this notion, we treated pregnant C3H/HeJ mice with daily oral native DTG at a human equivalent dosage (5 mg/kg; n = 6) or vehicle (control; n = 8). These were compared against pregnant mice injected with intramuscular (IM) NDTG formulations given at 45 (n = 3) or 25 (n = 4) mg/kg at one or two doses, respectively. Treatment began at gestation day (GD) 0.5. Magnetic resonance imaging scanning of live dams at GD 17.5 was performed to obtain T₁ maps of the embryo brain to assess T₁ relaxation times of drug-induced oxidative stress. Significantly lower T₁ values were noted in daily oral native DTG-treated mice, whereas comparative T₁ values were noted between control and NDTG-treated mice. This data reflected prevention of DTG-induced oxidative stress when delivered as NDTG. Proteomic profiling of embryo brain tissues harvested at GD 17.5 demonstrated reductions in oxidative stress, mitochondrial impairments, and amelioration of impaired neurogenesis and synaptogenesis in NDTG-treated mice. Pharmacokinetic (PK) tests determined that both daily oral native DTG and parenteral NDTG achieved clinically equivalent therapeutic plasma DTG levels in dams (4,000-6,500 ng/mL). Importantly, NDTG led to five-fold lower DTG concentrations in embryo brain tissues compared to daily oral administration. Altogether, our preliminary work suggests that long-acting drug delivery can limit DTG-linked neurodevelopmental deficits.

Keywords: HIV-1; dolutegravir; long-acting nanoformulations; neurodevelopment; pregnancy.

FIGURE 1

PK and BD profiles of NDTG during pregnancy. (A) Pre-mixture of dolutegravir (DTG) and Poloxamer 407 (P407) were subjected to homogenization using an Avestin EmulsiFlex-C3 high-pressure homogenizer to synthesize uniform NDTG particles. (B) NDTG particles were characterized for hydrodynamic particle diameter (size), polydispersity index (PDI), and zeta potential using a Malvern Zetasizer Nano-ZS. Drug content of formulation was measured using UPLC-TUV. (C) Morphological assessment of NDTG particles was performed using scanning electron microscopy (SEM). (D) Schematic representation of timeline and study design. Pregnant C3H/HeJ female mice (dams) were randomly distributed in four study groups. In group one, dams were daily administered native DTG at 5 mg/kg dose by oral gavage from gestation day (GD) 0.5 to GD 16.5. In group two, dams were daily administered with the vehicle by oral gavage from GD 0.5 to GD 16.5. In group three, dams were injected intramuscular (IM) with NDTG at 25 mg/kg dose on GD 0.5 and GD 16.5. In group four, dams were injected a single IM injection of NDTG at 45 mg/kg on GD 0.5. T₁ mapping, and embryo brain harvesting of DTG levels and proteomic assessments were completed on GD 17.5. (E) DTG concentrations in plasma of dams. DTG levels were measured at GD 16.5 to evaluate DTG levels in the mother’s blood. (F) DTG concentrations in whole brain tissues of embryos at GD 17.5. (G) DTG concentrations in placenta at GD 17.5. (E–G) Each sample (plasma or tissue) represents distinct litter. Data are expressed as mean ± SEM, N = minimum 3 animals/time point. t-test (two-tailed) was used (*p < 0.05, ^# p < 0.1).

FIGURE 2

T₁ mapping to examine T₁-relaxivity of oxidative stress in embryo brain. (A–E) T₁ mapping of embryo brain. Live dams were scanned at GD 17.5 to acquire T₁ maps to examine T₁-relaxivity of oxidative stress in embryo brain. T₁ mapping was performed using RARE with varying TR = 120–7,500 ms, and TE = 32 ms and in-house IDL program was used for T₁ fitting. Two embryos per dam were randomly selected for MRI scans. (A–D) Heat map and respective T₂-weighted image of representative embryo brain from each group are encompassed by red boxes. (E) Comparison of T₁ relaxation times among four study groups. N = minimum 6 animals/group. t-test (two-tailed) was used (*p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 3

Proteomic profiling of embryos brains following DTG or NDTG treatment. Non-targeted proteomic profiling of embryo whole brain tissues. Comparison between control and native DTG groups or control and NDTG groups were performed. Control: N = 7 animals; native DTG (oral): N = 9 animals; NDTG: N = 5 animals. (A,B) Volcano plot of significantly altered proteins after FDR correction. Red color: Upregulated proteins; Green color: Downregulated proteins; p ≤ 0.05; Absolute Fold Change ≥2 or ≤ −2. Schematic presentation of total number of affected proteins including upregulated and downregulated proteins. (C,D) Major affected canonical pathways were determined by using Ingenuity pathway analysis (IPA). Comparison was performed between (C) control and native DTG groups or (D) control and NDTG groups.

FIGURE 4

NDTG attenuates DTG-induced proteomic changes linked to mitochondrial dysfunction. Differentially expressed genes associated with mitochondrial dysfunction were determined by using IPA. Comparisons were performed between control and native DTG groups (red colored mitochondria, left side of the figure) or control and NDTG groups (green colored mitochondria, right side of the figure). Significantly differentially expressed genes were presented in heat maps. Control: N = 7 animals; native DTG (oral): N = 9 animals; NDTG: N = 5 animals.

FIGURE 5

Native DTG-linked developmental neuronal deficits. (A,B) Enrichment analysis of significantly altered proteins after FDR correction was completed using ShinyGo. Comparisons were performed between control and native DTG groups. DTG-exposure affected neuronal components were detected using GO-cellular components annotation. (C–E) Significantly differentially expressed genes associated with (C) synaptogenesis, (D) dendrite formation, and (E) neurogenesis were determined using IPA and presented in heat maps. Control: N = 7 animals; native DTG (oral): N = 9 animals.