Induction of oxidative stress by Taxol® vehicle Cremophor-EL triggers production of interleukin-8 by peripheral blood mononuclear cells through the mechanism not requiring de novo synthesis of mRNA

Abstract

Understanding the ability of cytotoxic oncology drugs, and their carriers and formulation excipients, to induce pro-inflammatory responses is important for establishing safe and efficacious formulations. Literature data about cytokine response induction by the traditional formulation of paclitaxel, Taxol®, are controversial, and no data are available about the pro-inflammatory profile of the nano-albumin formulation of this drug, Abraxane®. Herein, we demonstrate and explain the difference in the cytokine induction profile between Taxol® and Abraxane®, and describe a novel mechanism of cytokine induction by a nanosized excipient, Cremophor EL, which is not unique to Taxol® and is commonly used in the pharmaceutical industry for delivery of a wide variety of small molecular drugs.

From the clinical editor: Advances in nanotechnology have enabled the production of many nano-formulation drugs. The cellular response to drugs has been reported to be different between traditional and nano-formulations. In this article, the authors investigated and compared cytokine response induction profiles between Taxol® and Abraxane®. The findings here provided further understanding to create drugs with better safety profiles.

Keywords: Abraxane®; Cremophor-EL; Cytokines; Immunotoxicity; Interleukin 8; Oxidative stress; Paclitaxel; Taxol®.

Figure 1. Induction of pro-inflammatory cytokines by Taxol®, Abraxane® and Cremophor-EL in human whole blood

Whole blood derived from 6 healthy donor volunteers was left untreated or treated with designated agents for 20 h. The LPS (20 ng/mL) was used as the positive control (PC) and culture media was used as the negative control (NC). Culture supernatants were analyzed by ELISA for the presence of IL-8 (A, C), IL-1β, and TNF-α. Each bar represents the mean value of the duplicate sample obtained from individual donor (N=2, %CV < 20%). Reference to the individual donor (#1 through #6) is provided in the box shown on the right (B). Concentrations of Cremophor-EL in samples treated only with this vehicle were equivalent to those in Taxol® at respective concentrations of paclitaxel. These concentrations are referred to as μM of paclitaxel equivalent. Samples from each individual donor were analyzed in duplicate (N=2, %CV<20); each bar shows the mean response of 6 donors. Blood from the same donors as in (A) was used to generate this data. (C) Shown is the data obtained from one donor and analyzed in duplicate (N=2, %CV< 20); similar results were obtained from two other donors (Supplementary Figures 1B and 1C).

Figure 2. Effect of Cremophor-EL on paclitaxel-induced production of MIP-2 and TNF-α in murine macrophages

Raw 264.7 cells were incubated with test samples and controls for 20 h, and the secretion of MIP-2 (A) and TNF-α (B) was analyzed by ELISA. PC – positive control (20 ng/mL of the LPS); NC – negative control (culture medium); DMSO (5.9 mg/mL) was used as a vehicle control for paclitaxels; test samples were Taxol®, Abraxane®, and paclitaxel from different sources dissolved in DMSO. The effects of Cremophor-EL on paclitaxel-triggered MIP-2 (C) and TNF-α (D) secretion by murine macrophages were evaluated by simultaneous addition of Cremophor-EL and 12.5 μM of paclitaxel in DMSO to the cells. Shown is the mean response and standard deviation from three independent experiments (N=3). Each sample within individual experiment was analyzed in duplicate (%CV <20).

Figure 2. Effect of Cremophor-EL on paclitaxel-induced production of MIP-2 and TNF-α in murine macrophages

Raw 264.7 cells were incubated with test samples and controls for 20 h, and the secretion of MIP-2 (A) and TNF-α (B) was analyzed by ELISA. PC – positive control (20 ng/mL of the LPS); NC – negative control (culture medium); DMSO (5.9 mg/mL) was used as a vehicle control for paclitaxels; test samples were Taxol®, Abraxane®, and paclitaxel from different sources dissolved in DMSO. The effects of Cremophor-EL on paclitaxel-triggered MIP-2 (C) and TNF-α (D) secretion by murine macrophages were evaluated by simultaneous addition of Cremophor-EL and 12.5 μM of paclitaxel in DMSO to the cells. Shown is the mean response and standard deviation from three independent experiments (N=3). Each sample within individual experiment was analyzed in duplicate (%CV <20).

Figure 3. Secretion of IL-8 by human blood cells requires de novo synthesis of the protein but not mRNA

Human whole blood was treated with the negative control (NC, cell culture medium), the positive control (PC, 20 ng/mL of the LPS), or test agents (Taxol®, Cremophor-EL, or Abraxane®) at equivalent paclitaxel concentration of 25 μM. The amounts of IL-8 mRNA (A) and IL-8 protein (B) were measured in the same specimens; each bar represents mean response and standard deviation from ten donors (N=10). The level of IL-8 protein in supernatants 2 h (C) and 20 h (D) after treatment with Cremophor 25 μM and the PC. Each bar shown the mean response (N=2, %CV < 20) for each of 3 tested individual donors.

Figure 4. Cremophor-EL induces synthesis of the IL-8 protein de novo from pre-existing IL-8 mRNA

(A) Human PBMCs were treated with Taxol®, Abraxane®, or Cremophor-EL in the presence or absence of 5 μg/mL of protein synthesis inhibitor CHX. Supernatants were collected after 20 h of incubation and tested for the presence of IL-8 by ELISA. Shown is the mean response (N=2, %CV<20) from one donor; similar results were obtained from two more donors (Supplementary Figure 4A and B). (B) Northern blot analysis. PBMCs were left untreated or incubated with Cremophor-EL or the positive control (PC) for 4 h. The PC is 20 ng/mL of the LPS; Taxol® and Abraxane® were analyzed at equivalent paclitaxel concentrations of 25 μM; the concentration of Cremophor-EL used in this experiment was equivalent to what was used in Taxol® at 25 μM of paclitaxel. Shown is the representative data from one of three tested donors.

Figure 5. Cremophor-EL induces oxidative stress in human cells

Human PBMCs were treated with the negative control (NC), the positive control (PC), Taxol®, Abraxane®, or Cremophor-EL with or without 5 mM of NAC. (A) After 1 h of treatment, cells were loaded with fluorescent dye sensitive to oxidative stress and analyzed by flow cytometry. The shift in green fluorescent channel FL-1 intensity (X-axis) is indicative of oxidative stress. Carbonyl cyanide m-chlorophenylhydrazone was used as the PC to induce oxidative stress. Shown is representative data from one of 4 tested donors (B) The levels of the IL-8 protein were tested in culture supernatants by ELISA 20 h after treatment of cells from the same donors used in A. The NC is cell culture media and the PC is 20 ng/mL of the LPS; Taxol® and Abraxane® were tested at equivalent (25 μM) concentrations of paclitaxel. The concentration of Cremophor-EL in Cremophor-EL-treated samples was equivalent to that in Taxol® when the Taxol® was used at 25 μM of paclitaxel. Each bar represents the mean value of the duplicate sample obtained from individual donor (N=2, %CV < 20%). Reference to the individual donor (#1 through #4) is provided in the box shown on the right.

Figure 6. Effects of Cremophor-EL on mitochondrial potential

Human PBMCs were left untreated (A) or incubated with Cremophor-EL at two concentrations, 25 μM (B) and 12.5 μM (C), for 3 or 24 h. Shown is the representative data from one of three individual donors.

Figure 7. Effects of Cremophor-EL on peroxisome proliferation

Human PBMCs were treated with 12.5 μM of Cremophor-EL for 18 h prior to analysis of intracellular levels of PMP-70 by flow cytometry. Dotted line – isotype control; hatched filled histogram – NC; solid line – Cremophor-EL. Shown is the representative data from one of three individual donors.

Figure 8. Effects of Cremophor-EL on MAPK

(A) Human PBMCs were treated with 25 μM of Cremophor-EL and indicated concentrations of MAPK inhibitors. IL-8 was measured by ELISA in 20-h culture supernatants. Each bar represents the mean value of the duplicate sample obtained from individual donor (N=2, %CV < 20%). Reference to the individual donor (#1 through #3) is provided in the box shown on the right. Data are presented as the percentage of IL-8 protein induced by Cremophor-EL; the amounts of Cremophor-EL-triggered IL-8 were assigned to be 100%. (B) Human PBMCs were treated with 25 μM of Cremophor-EL for various time points before permeabilization and staining with antibodies specific to phosphorylated forms of p38 and Erk1/2. Dotted line – isotype control; filled histogram – NC; solid line – Cremophor-EL. Shown is the representative data from one of three individual donors.

Figure 8. Effects of Cremophor-EL on MAPK

(A) Human PBMCs were treated with 25 μM of Cremophor-EL and indicated concentrations of MAPK inhibitors. IL-8 was measured by ELISA in 20-h culture supernatants. Each bar represents the mean value of the duplicate sample obtained from individual donor (N=2, %CV < 20%). Reference to the individual donor (#1 through #3) is provided in the box shown on the right. Data are presented as the percentage of IL-8 protein induced by Cremophor-EL; the amounts of Cremophor-EL-triggered IL-8 were assigned to be 100%. (B) Human PBMCs were treated with 25 μM of Cremophor-EL for various time points before permeabilization and staining with antibodies specific to phosphorylated forms of p38 and Erk1/2. Dotted line – isotype control; filled histogram – NC; solid line – Cremophor-EL. Shown is the representative data from one of three individual donors.