Creation of a long-acting nanoformulated dolutegravir

Abstract

Potent antiretroviral activities and a barrier to viral resistance characterize the human immunodeficiency virus type one (HIV-1) integrase strand transfer inhibitor dolutegravir (DTG). Herein, a long-acting parenteral DTG was created through chemical modification to improve treatment outcomes. A hydrophobic and lipophilic modified DTG prodrug is encapsulated into poloxamer nanoformulations (NMDTG) and characterized by size, shape, polydispersity, and stability. Retained intracytoplasmic NMDTG particles release drug from macrophages and attenuate viral replication and spread of virus to CD4+ T cells. Pharmacokinetic tests in Balb/cJ mice show blood DTG levels at, or above, its inhibitory concentration₉₀ of 64 ng/mL for 56 days, and tissue DTG levels for 28 days. NMDTG protects humanized mice from parenteral challenge of the HIV-1_ADA strain for two weeks. These results are a first step towards producing a long-acting DTG for human use by affecting drug apparent half-life, cell and tissue drug penetration, and antiretroviral potency.

Fig. 1

Synthesis and characterization of MDTG. a A fourteen-carbon fatty-acid modified DTG prodrug (MDTG) was synthesized creating hydrophobic crystals at a final drug yield of 82.8%. b Absorption bands at 2915 cm⁻¹ and 2850 cm⁻¹ in the MDTG Fourier-transformation infrared spectrum (FTIR) illustrate the methyl C–H asymmetric and symmetric stretching of the myristic acid alkyl group. Bands at 1795 cm⁻¹ in the myristoyl chloride and 1750 cm⁻¹ in the MDTG FTIR spectrum correspond to carbonyl C = O stretching of the myristic acid acyl halide that reacts as the ester is formed in MDTG. c X-ray diffraction (XRD) analysis for DTG and MDTG demonstrates the crystalline structures of both drugs. d Aqueous solubility of DTG and MDTG demonstrates the decreased solubility of MDTG. ****P < 0.0001 DTG vs. MDTG. e IC₅₀ was determined in vitro in MDM by HIV-1 RT inhibition after DTG and MDTG treatment over a range of concentrations (0.01–1000 nM). Chemical modification of DTG did not affect antiretroviral drug activity (56.7 nM and 62.5 nM for DTG and MDTG, respectively; P = 0.8397). Results are shown as the mean ± SEM of three replicates. Results from d were analyzed by two-tailed Student’s t test (n = 10 DTG, 12 MDTG; t = 20.1, degrees of freedom = 20). Results from e were analyzed by nonlinear regression least squares fit

Fig. 2

Nanoformulation stability and release kinetics. Nanoformulations were synthesized by high-pressure homogenization using poloxamer 407 (P407) as the excipient for DTG and MDTG. a–c Formulation stability (up to 134 days) was measured by (a) particle hydrodynamic diameter (size), (b) polydispersity index (PDI), and (c) zeta potential as determined by dynamic light scattering (DLS). NDTG and NMDTG stability were tested at both 4 °C and 25 °C. d, e Nanoparticle release kinetics were assessed by drug released from the nanoparticles during storage, in (d) freshly manufactured (neat) formulation and (e) formulation diluted for in vivo administration. All results are shown as the mean ± SEM of at least three replicates

Fig. 3

Nanoparticle characterization. a, b Particle morphology was assessed by scanning electron microscopy (SEM). a Note that NDTG particles are of heterogeneous size and shape, and show both cuboidal and rod-shaped morphologies. b NMDTG particles are more uniform, with dominant rod-shaped morphologies (scale bars = 1 µm). c Drug uptake in MDM was measured over a 24-h period with equal drug concentrations (100 µM). Uptake of NMDTG was at or more than a half-log greater than its nonmodified control. ****P < 0.0001 NMDTG vs. NDTG. ^####P < 0.0001 MDTG vs. DTG. ^^P = 0.0054, ^^^^P < 0.0001 NMDTG vs. MDTG. d Drug retention in MDM was measured over a 30-day observation period demonstrating a log greater retention of the NMDTG compared to the nonmodified control. **P = 0.0048, ****P < 0.0001 NMDTG vs. NDTG. ^###P = 0.0004 MDTG vs. DTG. ^^^^P < 0.0001 NMDTG vs. MDTG. e Drug release from MDM was measured over a 14-day observation period demonstrating slowed and prolonged release from NMDTG-treated cells through 10 days. *P = 0.0156, ****P < 0.0001 NMDTG vs. NDTG. ^####P < 0.0001 MDTG vs. DTG. ^^^^P < 0.0001 NMDTG vs. MDTG. f Cell viability was assessed in MDM by MTT assay 24 h after NDTG or NMDTG treatment over a range of concentrations (1–500 µM). Results were normalized to untreated control cells. ****P < 0.0001 500 µM NDTG vs. control (untreated) cells. g–i Transmission electron microscopy (TEM) of (g) control, (h) NDTG, and (i) NMDTG loaded MDM after 8-h drug treatment. Note the paucity of particles in the NDTG-treated cells compared to the NMDTG-treated MDM (scale bars = 2 µm, upper panels; 500 nm, lower panels). Results are shown as the mean ± SEM of three biological replicates. Results from c, d, e, f were analyzed by two-way ANOVA with Bonferroni’s multiple comparison tests

Fig. 4

Antiretroviral efficacy. a, b HIV-1 RT activity of (a) DTG and MDTG, and (b) NDTG and NMDTG-treated MDM. **P = 0.0039, ***P = 0.0007, ****P < 0.0001 MDTG vs. DTG and NMDTG vs. NDTG. c Prevention of spreading viral infection was assessed in human peripheral blood lymphocytes (PBLs) following addition of media conditioned from drug-treated MDM. NMDTG conditioned media significantly reduced HIV-1 RT activity in PBLs compared to NDTG conditioned media beginning at day 15 and maintained protection up to day 24. **P = 0.0018, ****P < 0.0001 NMDTG vs. NDTG. d Representative HIV-1p24 staining (brown) of virus-infected MDM-treated with native or nanoformulated drugs are shown. For all, uninfected cells without treatment served as negative controls. HIV-1-infected cells without treatment served as positive controls. Results were normalized to positive control cells. All results are shown as the mean ± SEM of three biological replicates. Results from a, b, c were analyzed by two-way ANOVA with Bonferroni’s multiple comparison tests

Fig. 5

Pharmacokinetics. Balb/cJ mice were administered a single IM dose of NDTG or NMDTG (45 mg/kg DTG-eq.) to determine pharmacokinetic (PK) profiles. a Animal weights were monitored for the length of the study to assess animal health. b Blood DTG and MDTG concentrations were analyzed by UPLC-MS/MS. Solid lines indicate [DTG], while the dashed line indicates [MDTG] from NMDTG treatment. Dotted lines indicate the PA-IC₉₀ (64 ng/mL) and four-times the PA-IC₉₀ (256 ng/mL). c–h Tissue DTG concentrations were analyzed by UPLC-MS/MS. (c) Spleen, (d) lymph node, (e) GALT, (f) liver, (g) lung, and (h) kidney DTG levels are shown at days 28 and 56. ****P < 0.0001. Results are shown as the mean ± SEM of at least six biological replicates. Results from c–h were analyzed by two-tailed Student’s t test (for all, n = 6 NDTG, 6 NMDTG, degrees of freedom = 10; c t = 14.4; d t = 16.4; e t = 9.3; f t = 17.3; g t = 30.1; h t = 22.7)

Fig. 6

Protection against HIV-1 challenge in CD34+ humanized mice. CD34+ hematopoietic stem cell (HSC)- reconstituted NSG mice were treated with NMDTG according to the scheme illustrated in a. HIV-1-infected mice without treatment served as positive controls. b Blood DTG concentrations were analyzed by UPLC-MS/MS. Dotted lines indicate the PA-IC₉₀ (64 ng/mL) and four-times the PA-IC₉₀ (256 ng/mL). c DTG concentrations were also analyzed in spleen, GALT, lung, and liver samples. d Plasma viral load was measured three-weeks after HIV-1 challenge. e DNA and f RNA semi-nested real-time PCR was performed on spleen, GALT, lung, bone marrow, and liver. g HIV-1 RNAscope was performed on spleen and lymph node sections and scored according to amount of positive staining [0 = no staining or <1 dot/10 cells, 1 = 1–3 dots/cell, 2 = 4–9 dots/cell with no, or very few, dot clusters, 3 = 10–15 dots/cell and <10% dots are in clusters, 4 = >15 dots/cell and >10% dots are in clusters]. Anything scoring less than or equal to one was considered as background. h Representative HIV-1 RNAscope staining (brown) of spleen (top) and lymph node (bottom) sections are shown. *P < 0.05, **P < 0. 01. Results are shown as the mean ± SEM of five positive control (5 female) and 7 NMDTG-treated (5 female, 2 male) animals. Results were analyzed by two-tailed Student’s t test (n = 5 HIV-1 control, 7 MDTG; degrees of freedom = 10)