Development of a novel self-microemulsifying drug delivery system for reducing HIV protease inhibitor-induced intestinal epithelial barrier dysfunction

Abstract

The development of HIV protease inhibitors (PIs) has been one of the most significant advances of the past decade in controlling HIV infection. Unfortunately, the benefits of HIV PIs are compromised by serious side effects. One of the most frequent and deleterious side effects of HIV PIs is severe gastrointestinal (GI) disorders including mucosal erosions, epithelial barrier dysfunction, and leak-flux diarrhea, which occurs in 16-62% of patients on HIV PIs. Although the underlying mechanisms behind HIV PI-associated serious adverse side effects remain to be identified, our recent studies have shown that activation of endoplasmic reticulum (ER) stress response plays a critical role in HIV PI-induced GI complications. The objective of this study was to develop a novel self-microemulsifying drug delivery system (SMEDDS) using various antioxidants as surfactants and cosurfactants to reduce the GI side effects of the most commonly used HIV PI, ritonavir. The biological activities of this SMSDDS of ritonavir were compared with that of Norvir, which is currently used in the clinic. Rat normal intestinal epithelial cells (IEC-6) and mouse Raw 264.7 macrophages were used to examine the effect of new SMEDDS of ritonavir on activation of ER stress and oxidative stress. Sprague-Dawley rats and C57/BL6 mice were used for pharmacokinetic studies and in vivo studies. The intracellular and plasma drug concentrations were determined by HPLC analysis. Activation of ER stress was detected by Western blot analysis and secreted alkaline phosphatase (SEAP) reporter assay. Reactive oxygen species (ROS) was measured using dichlorodihydrofluorescein diacetate as a probe. Cell viability was determined by Roche's cell proliferation reagent WST-1. Protein levels of inflammatory cytokines (TNF-alpha and IL-6) were determined by enzyme-linked immunosorbent assays (ELISA). The intestinal permeability was assessed by luminal enteral administration of fluorescein isothiocyanate conjugated dextran (FITC-dextran, 4 kDa). The pathologic changes in intestine were determined by histological examination. The results indicated that incorporation of antioxidants in this new SMEDDS not only significantly reduced ritonavir-induced ER stress activation, ROS production and apoptosis in intestinal epithelial cells and macrophages, but also improved the solubility, stability and bioavailability of ritonavir, and significantly reduced ritonavir-induced disruption of intestinal barrier function in vivo. In conclusion, this new SMEDDS of ritonavir has less GI side effects compared to Norvir. This new SMEDDS can be used for other HIV PIs and any insoluble antiviral drug with serious GI side effects.

Figure 1. Effect of antioxidants on ritonavir-induced ROS production in IEC-6 cells

Cells were treated with vehicle control (DMSO), positive control (H₂O₂, 10 µM), thapsigargin (TG, a known ER stress inducer, 100 nM), ritonavir (RTV, 15 µM), RTV with vitamin C (VC 15 µg/ml) or, vitamin E (VE 20 µg/ml), or ethyl linoleate (EL, 20 µg/ml) or oleic acid (OA, 10 µg/ml) for 4h. The intracellular ROS levels were measured as described in “Methods”. Values are mean ± SD of 3 independent experiments and analyzed using one-way ANOVA, Statistical significance relative to control, *p < 0.05 or relative to RTV, #p<0.05.

Figure 2. Effect of antioxidants on ritonavir-induced ER stress and UPR activation in IEC-6 cells

A. IEC-6 cells stably transfected with pSEAP plasmid were treated with vehicle control (DMSO), ritonavir (RTV, 15 µM) in the absence or presence of individual antioxidant, vitamin C (VC 15 µg/ml) or, vitamin E (VE 20 µg/ml), or ethyl linoleate (EL, 20 µg/ml) or oleic acid (OA, 10 µg/ml) or TG (100 nM) for 24 h. Activity of SEAP was measured as described under “Methods” and expressed as percent of control. Values are mean ± SD of 3 independent experiments. Statistical significance relative to vehicle control, * p< 0.05 or relative to RTV, #p<0.05. B. Representative immunoblots against CHOP and lamin B from the nuclear extracts of IEC-6 cells treated with ritonavir (RTV, 15 µM) in the absence or presence of individual antioxidant, vitamin C (VC 15 µg/ml) or, vitamin E (VE 20 µg/ml), or ethyl linoleate (EL, 20 µg/ml or 40 µg/ml) or oleic acid (OA, 10 µg/ml) for 4 h. Lamin B was used as a loading control of nuclear protein. Relative protein levels of CHOP from three independent experiments. Statistical significance relative to control, *p < 0.05 or relative to RTV, #p<0.05.

Figure 3. Characterization of ritonavir SMEDDS

A: Particle size distribution of SMEDDS in water. 1: blank SMEDDS 46.1 nm; 2: RTV SMEDDS 81.6 nm. B–C: Zeta potential of SMEDDS, B: blank SMEDDS 1.94 mv; C: RTV SMEDDS 1.70 mv.

Figure 4. Stability and pharmacokinetics of ritonavir SMEDDS

A. The stability of ritonavir SMEDDS (RTV ME), and Norvir® in medium. RTV ME and Norvir® (25 µM) were incubated in cell culture medium at 37°C for 4, 24 and 48 h. The remaining ritonavir in medium was determined by HPLC as described in “Methods”. B. IEC-6 cells were treated with 15 µM ritonavir SMEDDS (RTV ME), Norvir® or ritonavir in DMSO (RTV DMSO) for 0.25, 0.5, 1, 2, 4, 8 and 24 h. The intracellular ritonavir concentration was determined by HPLC as described in “Methods” and normalized to total protein amount. Each data represents mean ± S.E. of three experiments. C. The in vivo Pharmacokinetics of RTV ME. Sprague-Dawley rats were gavaged with RTV ME or Norvir® at a dose of 50 mg/kg. The plasma concentration of ritonavir was determined as described in “Methods”.

Figure 5. Cell viability assay in IEC-6 cells

IEC-6 cells were seeded on 96-well plates for overnight, then treated with 25 µM of ritonavir SMEDDS (RTV ME), Norvir®, or ritonavir in DMSO (RTV DMSO) for 24 h. The cell viability was determined using Cell Proliferation Reagent WST-1 as described in “Methods”. Each data represents mean ± SD. of three experiments. Statistical significance relative to control, *p < 0.05 or relative to RTV, #p<0.05.

Figure 6. Effect of ritonavir SMEDDS and Norvir® on ROS production in IEC-6 cells

Cells were treated with vehicle control (DMSO), positive control (H₂O₂, 10 µM), ritonavir in DMSO (RTV DMSO, 15 µM), blank SMEDDS (blank ME), ritonavir SMEDDS (RTV ME, 15 µM), and Norvir® (15 µM) for 4h. The intracellular ROS levels were determined using H2DCFDA as described in “Methods”. Each bar represents mean ± SD of three experiments. Statistical significance relative to DMSO control,*p < 0.05 and relative to RTV DMSO, #p<0.05.

Figure 7. Effect of ritonavir SMEDDS on ER stress and UPR activation in IEC-6 cells

A. IEC-6 cells stably transfected with pSEAP plasmid were treated with vehicle control (DMSO), ritonavir in DMSO (RTV DMSO, 15 µM), ritonavir SMEDDS (RTV ME, 15 µM) or Norvir® (15 µM), or TG (25 nM) for 24 h. Activity of SEAP was measured as described under “Methods” and expressed as percent of control. Values are mean ± SD of 3 independent experiments. Statistical significance relative to vehicle control, * p< 0.05, or relative to RTV DMSO, #p<0.05, B. Representative immunoblots against CHOP and lamin B from the nuclear extracts of IEC-6 cells treated with vehicle control (DMSO), ritonavir in DMSO (RTV DMSO, 15 µM), ritonavir SMEDDS (RTV ME, 15 µM) or Norvir® (15 µM), or TG (25 nM) for 4 h. Lamin B was used as a loading control of nuclear protein. Relative protein levels of CHOP from three independent experiments were analyzed. Statistical significance relative to control, *p < 0.05 or relative to RTV DMSO, #p<0.05.

Figure 8. Effect of ritonavir SMEDDS on intestinal permeability and tissue damages in vivo

Wild type C57BL/6 mice were treated with control solution (PBS), blank SMEDDS(Blank ME), ritonavir SMEDDS (RTV ME) or Norvir® (50 mg/kg) for 4 weeks. A. The intestinal permeability was measured using FITC-dextran as described in “Methods”. Statistical significance relative to vehicle control (n=5), *p<0.05, **p<0.01. B. Representative images of HE staining for each treatment group are shown.