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Anti-inflammatory and cytoprotective effects of a squalene synthase inhibitor, TAK-475 active metabolite-I, in immune cells simulating mevalonate kinase deficiency (MKD)-like condition
© The Author(s) 2016
Received: 9 February 2016
Accepted: 23 August 2016
Published: 30 August 2016
TAK-475 (lapaquistat acetate) and its active metabolite-I (TAK-475 M-I) inhibit squalene synthase, which catalyzes the conversion of farnesyl diphosphate (FPP) to squalene. FPP is a substrate for synthesis of other mevalonate-derived isoprenoids (MDIs) such as farnesol (FOH), geranlygeranyl diphosphate (GGPP), and geranylgeraniol. In patients with MKD, a rare autosomal recessive disorder, defective activity of mevalonate kinase leads to a shortage of MDIs. MDIs especially GGPP are required for prenylation of proteins, which is a posttranslation modification necessary for proper functioning of proteins like small guanosine triphosphatases. Malfunction of prenylation of proteins results in upregulation of the inflammatory cascade, leading to increased production of proinflammatory cytokines like interleukin-1β (IL-1β), eventually leading to episodic febrile attacks. In vitro, TAK-475 M-I incubation in a concentration dependent manner increased levels of FPP, GGPP, and FOH in human monocytic THP-1 cells. In subsequent experiments, THP-1 cells or human peripheral blood mononuclear cells (PBMCs) were incubated with simvastatin, which inhibits hydroxymethylglutaryl-coenzyme A reductase and thereby decreases levels of the precursors of MDIs, leading to the depletion of MDIs as expected in MKD patients. Increased levels of GGPP and FPP attenuated lipopolysaccharide (LPS)-induced IL-1β production in THP-1 cells and human PBMCs in statin-treated conditions. The MDIs also significantly reduced the damaged cell ratio in this active MKD-like condition. Moreover, TAK-475 M-I directly inhibited LPS-induced IL-1β production from statin-treated THP-1 cells. These results show anti-inflammatory and cytoprotective effects of MDIs via TAK-475 M-I treatment in statin-treated immune cells, suggesting that possible therapeutic effects of TAK-475 treatment in MKD patients.
Mevalonate kinase deficiency (MKD) is a rare periodic fever syndrome with autosomal recessive inheritance that is caused by a mutation in the MVK gene encoding mevalonate kinase (MK) (Esposito et al. 2014). MKD patients suffer from autoinflammatory disorders characterized by recurrent episodes of fever (Bader-Meunier et al. 2011) accompanied by painful lymphadenopathy; gastrointestinal symptoms, and so on (van der Hilst et al. 2008; van der Burgh et al. 2013; Ter Haar et al. 2013; Lainka et al. 2012). The MK gene mutation causes a dramatic decrease in MK enzymatic activity. MK converts mevalonic acid to mevalonate-5 phosphate, which is the precursor for the synthesis of a range of MDIs, including FPP, FOH, GGPP, GGOH, ubiquinones, and dicarboxylic acids (DCAs) (van der Burgh et al. 2013; Tricarico et al. 2014; Hubner et al. 1993; Xu et al. 2015). Consequently, MDI levels in MKD patients are reported to be decreased (Hubner et al. 1993). MDIs play a crucial role in the regulation and production of a range of cytokines, and decreased MDI levels are implicated in increases in pro-inflammatory mediators, including interleukin-1β (IL-1β), interleukin-6, C-reactive protein (CRP), and monocyte chemotactic protein-1 (Kostjukovits et al. 2015; Mandey et al. 2006; Marcuzzi et al. 2013). Moreover, an increased production of inflammatory cytokines by peripheral blood mononuclear cells (PBMCs), including monocytes, has been suggested as a central mechanism in the inflammatory phenotype of MKD.
TAK-475 M-I (T-0091485, Lot No. X000000090965) was synthesized in Chemical Development Laboratories of Takeda Pharmaceutical Company Limited. For internal control, GGPP (Lot No. E00166-66) and FPP (Lot No. MJB-E00026-28) were purchased from Echelon Biosciences. GGOH (Lot No. 2598758) was purchased from LKT Laboratories. FOH (Lot No. MKBK0393 V) was purchased from Sigma-Aldrich. LPS (Lot No. PDM3922) were purchased from Wako Pure Chemical Industries.
Cell cultures and treatments
Human PBMCs isolation and treatment
Measurements of the concentration of MDIs
FOH, FPP, GGOH and GGPP in cells were quantified by LC–MS/MS by Sumika Chemical Analysis Service., and the lowest detection limit was 0.008 μg/1.5 × 106 cells, and the highest detection limit was 2 μg/1.5 × 106 cells. Values below the detection limit were presumed to be 0.008 μg/1.5 × 106 cells, and values higher the detection limit were presumed to be 2 μg/1.5 × 106 cells.
Extraction and enzymatic hydrolysis
In cell samples (1.5 × 106 cells), 100 μL of 8 % KOH ethanol solution was added and the samples were incubated at 80 °C for 2 h. The samples were agitated during the incubation to disintegrate the plasma and cell efficiently. The obtained solution was cooled down to ambient temperature and centrifuged at 3000 rpm for 5 min. To the obtained supernatant (50 μL), 10 μL of n-pentadecanol (I.S.) solution (1 μg/mL) and 2 mL of 1.5 mol/L Tris–HCl buffer (pH8.6) containing 0.25 mol/L MgCl2 were added. FOH, GGOH and I.S. in the solution were extracted with 5 mL of n-hexane/ethanol (98.5:1.5, v/v, 2 times), and the obtained supernatant by centrifugation at 3000 rpm for 5 min was evaporated under a stream of N2 gas. FPP and GGPP were quantified as FOH and GGOH, respectively (Ctotal) together with free FOH and GGOH (Cfree) after enzymatic hydrolysis using alkaline phosphatase. The concentrations of FPP and GGPP were calculated by subtracting the Cfree from Ctotal. If the calculated value is negative, the concentration is described as “0.00”. For enzymatic hydrolysis, alkaline phosphatase (1 unit) were added to 8 % KOH sample solution (plasma: 20 μL, cell: 50 μL) with 2 mL of 1.5 mol/L Tris–HCl buffer (pH 8.6) containing 0.25 mol/L MgCl2, and samples were incubated at 37 °C for 2 h. The following extraction procedure was the same as described above.
Derivatization of FOH and GGOH
To above extracts, 250 μL of 3-nitrophthalic anhydride pyridine solution (10 mg/mL) was added, and the samples were incubated at 70 °C for 30 min. After the incubation, pyridine was evaporated under a stream of N2 gas. The residue was dissolved in 200 μL of methanol, and 500 μL of 50 mmol/L phosphate buffer (pH3) was added to the methanol solution. The derivatives of FOH, GGOH and I.S. (FOH-NPA, GGOH-NPA and I.S.-NPA respectively) in the solution were extracted with 5 mL of n-hexane. n-hexane extract was concentrated under the N2 gas, the residue was dissolved in a mixture of methanol/water/acetic acid (75:25:0.1, v/v/v) (200 μL) for LC–MS/MS analysis. A 10 μL portion of the solution was injected into the LC–MS/MS system.
Peaks on the chromatogram were identified based on the retention time and the mass-to-charge ratio (m/z) of the monitoring ions. The concentrations of FOH and GGOH were determined from the peak area of FOH and GGOH using the absolute calibration curve method because I.S. peak area was varied widely. The calibration curves of FOH and GGOH were linear in the spiked concentration range from 0.008 to 0.8 μg/mL.
Measurements of IL-1β protein concentration by ELISA and statistics
Cytotoxicity assay and statistics
Cytotoxicity assay was conducted using ToxiLight bioassay kit (Lonza, Basel, Switzerland), a bioluminescent, non-destructive cytolysis assay kit designed to measure the release of the enzyme, adenylate kinase (AK), from damaged cells. Damaged cell ratio (DCR) was estimated by dividing AK activity from AK-released cells by that from total cells. Data are represented as the mean + SD (n = 3). The cytotoxicity of LPS was evaluated by a Student’s t test, and the dose-dependent effects of MDIs in LPS-stimulated MKD-like THP-1 cells were evaluated by a two-tailed Williams’ test. In both tests, P < 0.05 was considered statistically significant (Fig. 3). The cytotoxicity of simvastatin and LPS in THP-1 cells or the effects of GGPP and FPP in LPS-stimulated MKD-like THP-1 cells were evaluated by a Dunnett’s test. P < 0.05 was considered statistically significant (Fig. 5).
TAK-475 M-I significantly increased the levels of MDIs in THP-1 cells
Effects of TAK-475 M-I on the level of four MDIs; FPP, FOH, GGPP and GGOH in human monocytic THP-1 cells were examined. THP-1 cells were treated for 48 h with 0, 30, 100, 150, 300 and 1000 nmol/L of TAK-475 M-I. The concentrations of MDIs were calculated assuming that 106 THP-1 cell volume is equal to 970 μL (Kuijk et al. 2008). Levels of FPP and GGPP were increased by 48 h in a dose-dependent manner, whereas GGOH levels did not show any apparent changes (Fig. 1). Although there is a poor dose-dependency in FOH induction, a trend is observed in that high concentration of TAK-475 M-I can induce more FOH (Fig. 1). TAK-475 M-I potently inhibited de novo cholesterol synthesis in human primary hepatocytes with relative IC50 values of 110 nmol/L (unpublished data). It should be obviously difficult to detect cholesterol level in human monocytic THP-1 cells compared with human hepatocytes because cholesterol is mainly synthesized in the liver. If TAK-475 M-I can inhibit cholesterol synthesis in THP-1 cells like in human primary hepatocytes, TAK-475 M-I is suggested to be able to strongly increase FPP, FOH and GGPP with the effect against cholesterol inhibition.
MDIs blocked LPS-stimulated IL-1β production in MKD-like THP-1 cells
The mean IC50 of MDIs for the inhibition of LPS-stimulated IL-1β production in MKD-like THP-1 cells
95 % confidence interval
MDIs also reduced cytotoxicity in LPS-stimulated MKD-like THP-1 cells
Effects of MDIs on cytotoxicity in LPS-stimulated MKD-like human monocytic THP-1 cells were investigated. The treatment of 10 ng/mL of LPS increased DCR from approximately 20–35 % (Fig. 3). MDIs showed inhibitory effects against LPS-induced cytotoxicity in statin-treated THP-1 cells (Fig. 3). GGPP reduced LPS-induced DCR by approximately 70 % even at 1 μmol/L concentration, because 35 % DCR became 10 % DCR by shaving off 25 % worth of cytotoxicity which is about 70 % of LPS-induced DCR. FPP also reduced LPS-induced DCR by approximately 70 % at 10 μmol/L concentration. Moreover, other MDIs such as GGOH and FOH can also significantly reduce LPS-induced cytotoxicity in MKD-like condition (Fig. 3).
MDIs blocked LPS-stimulated IL-1β production in MKD-like human PBMCs
Effects of GGPP and FPP on LPS-stimulated IL-1β production in statin-treated human PBMCs were investigated. Human PBMCs were pretreated with simvastatin (3 μmol/L) for 24 h to induce an MKD-like phenotype by reducing endogenous MDI levels (Kuijk et al. 2008), and then stimulated with LPS (200 ng/mL) in the presence of GGPP or FPP (10 μmol/L) for an additional 24 h. Both GGPP and FPP at 10 μmol/L significantly inhibited LPS-stimulated IL-1β production in MKD-like human PBMCs in all 3 donors (Fig. 4). It was obvious that GGPP had a much stronger inhibitory effect compared with FPP, confirming the IC50 value on Table 1 (Fig. 4).
MDIs also showed a tendency to reduce cytotoxicity in LPS-stimulated MKD-like human PBMCs
DCR of PBMCs increased with the treatment of simvastatin and LPS (Fig. 5). However, GGPP or FPP (10 μmol/L) showed a tendency to decrease the DCR in all donors. Judging from Figs. 4 and 5, both GGPP and FPP at 10 μmol/L significantly inhibited LPS-stimulated IL-1β production without cytotoxicity in MKD-like human PBMCs.
TAK-475 M-I suppresses LPS-stimulated IL-1β production in both MKD-like THP-1 cells and human PBMCs
TAK-475 and its active metabolite M-I inhibit the enzyme that catalyzes the conversion of FPP to squalene on the cholesterol biosynthesis pathway. TAK-475 has been evaluated extensively in both in vitro and in vivo experimental models that have confirmed cholesterol-lowering properties (Nishimoto et al. 2003; Amano et al. 2003). In patients with MKD, defective activity of the MK enzyme is hypothesized to lead to a shortage of MDIs such as FPP. Through squalene synthase inhibition in the cholesterol biosynthesis pathway, TAK-475 disrupts the conversion of FPP into squalene resulting in increased levels of FPP and other MDIs.
The effects of TAK-475 M-I on MDIs were examined in in vitro experiments. These results showed that treatment with TAK-475 M-I resulted in the significant increase of MDIs such as FPP, FOH, and GGPP in human monocytic THP-1 cell line, as shown in Fig. 1. In subsequent studies potential inhibitory effects of MDIs on IL-1β secretion were investigated in MKD-like conditions. THP-1 cells and PBMCs were first incubated for 24 h with simvastatin, which inhibits hydroxymethylglutaryl-coenzyme A (HMG-CoA) and thereby decreases levels of the precursors for MDI production (Bellosta et al. 2000), leading to MDI depletion as expected in MKD patients. The cells were then stimulated for 24 h with LPS, which induces IL-1β secretion, while in the presence of MDIs. Results from this study demonstrated that pretreatment of FPP, GGPP, and GGOH in a concentration-dependent manner decreased LPS- induced IL-1β secretion in MKD-like THP-1 cell lines (Fig. 2). Similarly, GGPP and FPP both decreased LPS-induced IL-1β secretion in MKD-like human PBMCs regardless of the donor’s source (Fig. 4). The production of inflammatory cytokines such as IL-1β is considered as one of the causes of fever episodes in MKD patients (Galeotti et al. 2012). The increased levels of MDIs after treatment with TAK-475 are therefore expected to mitigate the severity and/or frequency of fever episodes in patients with MKD.
According to the human plasma PK profile of TAK-475, oral administration of TAK-475 100 mg once daily leads to the average plasma concentration of approximately 30 nmol/L of total active concentration of TAK-475 and its active metabolites (data not shown). Therefore, based on the detailed data in Fig. 1, it is anticipated that 30 nmol/L of TAK-475 can induce approximately 427 µmol/L of FPP and approximately 88 µmol/L of GGPP in THP-1 cells. The anticipated increase in levels of MDIs may be much less in MKD patients compared to healthy human subjects because of very limited availability of isopentenyl pyrophosphate (IPP). However, given that the mean inhibitory concentrations (IC50) of FPP and GGPP are very low at 1.18 µmol/L and 1.5 nmol/L, respectively, in MKD-like THP-1 cells (Table 1), a slight increase in FPP and GGPP levels induced by TAK-475 100 mg may give sufficient inhibition of IL-1β production in MKD patients and mitigate fever episodes. Moreover, treatment with TAK-475 M-I was confirmed to inhibit IL-1β production in THP-1 cell lines exhibiting an MKD-like phenotype in 35 % reduction at 150 nmol/L and 23 % reduction at 1000 nmol/L (Fig. 6). It is also confirmed that TAK-475 M-I can inhibit IL-1β production even in MKD-like human PBMCs in 41 % reduction at 1000 nmol/L (Fig. 7). Increased MDIs by TAK-475 treatment would be able to block the production of inflammatory cytokines, which is one of the causes of fever episodes in MKD patients, in the clinical setting (Marcuzzi et al. 2013; Galeotti et al. 2012). Similarly, Kuijk et al. (2008) showed that the addition of GGPP led to a decrease in levels of IL-1β in THP-1 cell lines that were treated with simvastatin to produce MKD-like inflammatory phenotype.
Interestingly, MDIs have significant cytoprotective effects in LPS-stimulated THP-1 cells, which are treated with simvastatin to induce an MKD-like phenotype. It is reported that statins induce apoptosis via caspase-3 activation in glioma cells (Yanae et al. 2011). As LPS stimulation is also known to induce apoptosis in some endothelial cells, it would be reasonable that LPS treatment enhanced DCR 75 % more in MKD-like THP-1 cells by changing from 20 % DCR into 35 % DCR (Fig. 3). This DCR was most significantly inhibited by GGPP as shown Fig. 3. This might be due to the inhibition caspase-3 activity via ERK1/2 and Akt activations because the supplementation of MDIs such as GGPP is reported to enhance both Ras-ERK1/2 and Ras-Akt signaling pathways (Yanae et al. 2011). Similarly, the supplementation of both GGPP and FPP decreased LPS-induced DCR in statin-treated human PBMCs (Fig. 5). These results suggested that TAK-475 could recover the damaged cells of MKD patients, which were caused by the decreases levels of the precursors for MDI production.
Our results suggest that TAK-475 M-I can block the production of inflammatory cytokine IL-1β as well as significantly enhanced the cytoprotective effect in MKD-like human immune cells, indicating that TAK-475 has a potential to be the disease modifying drug in patients with MKD.
In this study, we have demonstrated the anti-inflammatory and cytoprotective potentials of TAK-475 M-I, benzoxazepine derivative SSI, through the accumulation of MDIs in immune cells simulating MKD-like condition. As the shortage of MDIs could be the main cause of MKD symptoms such as fever episodes, the disease-modifying therapeutic effects will be expected by TAK-475 treatment in MKD patients.
Conceived and designed the experiments: NS TI HM MT. Performed the experiments: NS TI. Analyzed the data: NS TI HM MT. Contributed reagents/materials/analysis tools: NS TI. Wrote the paper: NS. All authors read and approved the final manuscript.
We are grateful to Drs. Michie Hisada and Saurabh Gupta for critical reading of the manuscript and useful feedbacks. We thank Drs. Tauhid Ali, Richard Philipson, Guido Hanauer, Takuya Ebihara, Shotaro Miura, Hideo Fukui, Kok Wah Hew, Katsunori Takashima, Taku Sugita, Masatoshi Karashima, and Fumihiro Jinno for fruitful discussions. Special thanks go to Dr. Ryuichi Tozawa for help and useful suggestions.
The authors declare that they have no competing interests.
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