Optimizing the preparation and stability of decorated antiretroviral drug nanocrystals

Abstract

Aim: While the therapeutic potential for current long-acting (LA) antiretroviral therapy (ART) is undeniable, ligand-decorated nanoformulated LA-ART could optimize drug delivery to viral reservoirs. The development of decorated ART hinges, however, on formulation processes and manufacture efficiencies. To this end, we compared manufacture and purification techniques for ligand-decorated antiretroviral drug nanocrystals.

Materials & methods: Ligand-decorated nanoparticle manufacturing was tested using folic acid (FA) nanoformulated cabotegravir.

Results: Direct manufacturing of FA-cabotegravir resulted in stable particles with high drug loading and monocyte-macrophage targeting. A one step 'direct' scheme proved superior over differential centrifugation or tangential flow filtration facilitating particle stability and preparation simplicity and efficiency.

Conclusion: Direct manufacturing of FA nanoparticles provides a path toward large-scale clinical grade manufacturing of cell-targeted LA-ART.

Keywords: cabotegravir; long-acting antiretrovirals; monocyte-derived macrophage; nanocrystals; uptake and release.

Figure 1.. Chemical structure of cabotegravir.

Cabotegravir is a hydrophobic integrase inhibitor under development for treatment and prevention of HIV infection. It has a long half-life of 21–50 days after a single parenteral dose.

Figure 2.. Size reduction scheme for folic acid nanoformulated cabotegravir.

1% (w/v) cabotegravir was dispersed in polymer solutioncontaining both targeted and nontargeted P407 polymer, followed by high-pressure homogenization until the desired particle size and polydispersity index was achieved. The size-reduced particles contained excessive polymers and needed further purification. CAB: Cabotegravir; FA: Folic acid; FA-P407: FA conjugated poloxamer 407; P407: Poloxamer 407.

Figure 3.. Folic acid nanoformulated cabotegravir preparations purified by differential centrifugation

(A) Post homogenization process scheme of FA NCAB purified by differential centrifugation. High speed centrifugation at 10,000 × g was used to pellet FA NCAB followed by resuspension in 0.2% (w/v) P407 solution. Large particles that cannot disperse during resuspension were removed by low speed centrifugation at 200 × g. (B) Morphology of FA NCAB purified by centrifugation was visualized by scanning electron microscopy. Scale bar: 5 μm. (C) Cellular uptake of FA NCAB prepared by differential centrifugation was assessed in human MDM and compared with nanoformulation without targeting ligand (NCAB). Intracellular drug concentrations were determined in MDM treated with 100 μM NCAB or FA NCAB for 1–8 h. Intracellular drug concentrations were analyzed by HPLC-UV/vis. Data are expressed as mean ± standard deviation for n = 3 samples per group. For each time point, means were compared by two-tailed Student's t-test. *p < 0.05; **p < 0.01; ***p < 0.001. CAB: Cabotegravir; FA NCAB: Folic acid decorated nanoformulated CAB; FA-P407: FA conjugated poloxamer 407; MDM: Monocyte-derived macrophage; P407: Poloxamer 407.

Figure 4.. Folic acid decorated nanoformulated cabotegravir preparations by tangential flow filtration.

(A) Manufacturing scheme for FA NCAB purified by tangential flow filtration (TFF). FA NCAB was purified by TFF diafiltration using 5 or 10 DV of water. (B) Time course measurements of FA NCAB prepared by TFF for Deff, ζ-potential and polydispersity index. Data are expressed as mean ± standard deviation for n = 3 measurements. (C) Morphology of FA NCAB purified by TFF was visualized by scanning electron microscopy. Scale bar: 5 μm. (D) Cellular uptake of FA NCAB prepared by TFF was assessed in human MDM and compared with formulation without targeting ligand or NCAB. Intracellular drug concentrations were determined in MDM treated with 100 μM NCAB or FA NCAB for 2 and 4 h. Intracellular drug concentrations were analyzed by HPLC-UV/vis. Data are expressed as mean ± standard deviation for n = 3 samples per group. One-way ANOVA followed by Tukey's post hoc test was used to compare FA NCAB with nontargeted NCAB formulation. ***p < 0.001. CAB: Cabotegravir; DV: Diavolume; FA NCAB: Folic acid decorated nanoformulated cabotegravir; MDM: Monocyte-derived macrophage; PdI: Polydispersity index.

Figure 5.. Proton nuclear magnetic resonance spectrum of lyophilized folic acid decorated nanoformulated cabotegravir formulation purified by tangential flow filtration.

Drug to P407 ratio was calculated based on characteristic integral peak areas of CAB and P407. CAB: Cabotegravir; ¹H-NMR: Proton nuclear magnetic resonance; P407: Poloxamer 407; TFF: Tangential flow filtration.

Figure 6.. Direct preparation of folic acid decorated nanoformulated cabotegravir.

(A) Manufacturing scheme of FA NCAB prepared by a direct method. (B) Time course measurements of direct prepared FA NCAB over 120 days for Deff, ζ-potential and polydispersity index. Data are expressed as mean ± standard deviation (SD) for n = 3 measurements. (C) Morphology of FA NCAB prepared by the direct method was visualized by scanning electron microscopy. Scale bar: 5 μm. (D) Monocyte-derived macrophage (MDM) uptake of NCAB and FA NCAB prepared by the direct method. MDM were treated with 100 μM NCAB or FA NCAB, and intracellular drug concentrations were analyzed by HPLC-UV/vis at 1, 2 and 4 h after drug treatment. Data are expressed as mean ± SD for n = 3 samples. For each time point, means were compared by two-tailed Student's t-test. **p < 0.01; ***p < 0.001. (E) Competitive uptake study of FA NCAB. Cells were pretreated with free FA (0–25 mM) for 30 min to block folate receptors, followed by FA NCAB treatment. Data are expressed as mean ± SD for n = 3 samples. CAB: Cabotegravir; FA: Folic acid; FA NCAB: Folic acid decorated nanoformulated cabotegravir; FA-P407: FA conjugated poloxamer 407; PdI: Polydispersity index.

Figure 7.. Folic acid decorated nanoformulated cabotegravir pharmacokinetic and biodistribution profiles.

Male BALB/cJ mice were administered intramuscularly 45 mg/kg FA NCAB purified by TFF or directly prepared to 45 mg/kg CAB-long-acting parenteral treatment. (A) Plasma was collected at days 3, 7, 14, 21 and 28 after drug administration and CAB concentrations were determined by ultra performance liquid chromatography tandem mass spectrometry. Data represent mean ± standard error of the mean for n = 5 mice per group. (B) Tissues were collected at day 28 and CAB concentrations were determined by ultra performance liquid chromatography tandem mass spectrometry. Data are expressed as mean ± standard error of the mean for n = 5 mice per group. Tissue CAB concentrations were compared by one-way ANOVA followed by Tukey's post hoc test. *p < 0.05; **p < 0.01. CAB: Cabotegravir; CAB-LAP: Cabotegravir long-acting parenteral; FA NCAB: Folic acid decorated nanoformulated cabotegravir; TFF: Tangential flow filtration.