Journal of Immunology, Infection & Inflammatory Diseases

Research Article

Solithromycin Diminishes Steatohepatitis by Modulating Gluconeogenesis and Inhibits Tumor Growth in a Diabetic Mouse Model of Non-Alcoholic Steatohepatitis

Prabhavathi Fernandes, David Oldach, Taishi Hashiguchi, Yuka Shirakata, Hiroyuki Yoneyama, Pierre M. Gholam

Correspondence Address :

Prabhavathi Fernandes
Cempra, Inc. 6320 Quadrangle Drive
Building Two
Suite 360
Chapel Hill
NC 27517
USA
Tel: 919-313-6610
Email: pfernandes@cempra.com

Received on: June 22, 2016, Accepted on: July 08, 2016, Published on: July 12, 2016

Citation: Prabhavathi Fernandes, David Oldach, Taishi Hashiguchi, Yuka Shirakata, Hiroyuki Yoneyama, Pierre M. Gholam (2016). Solithromycin Diminishes Steatohepatitis by Modulating Gluconeogenesis and Inhibits Tumor Growth in a Diabetic Mouse Model of Non-Alcoholic Steatohepatitis

Copyright: 2016 Fernandes P, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

New treatments are needed for patients with Non-Alcoholic Steatohepatitis (NASH). We examined the effects of solithromycin, a novel macrolide antibiotic, on steatohepatitis, fibrosis and Hepatocellular Carcinoma (HCC) in a NASH-HCC mouse model. NASH was induced in 2-day old mice by streptozotocin injection and a high fat diet. NASH mice were treated with oral solithromycin (i) between 4-8 weeks to examine the effect on NASH or (ii) between 8-12 weeks to examine the effect on fibrosis or (iii) between 4-20 weeks to determine the effect on the development of HCC. Non-fasting blood glucose, serum and liver triglyceride and lipid concentrations were measured. NAFLD Activity Score (NAS) and the grade of fibrosis were investigated by histopathology. Relative mRNA expression levels in the liver were determined for a variety of marker genes. At week 8 (NASH phase), solithromycin significantly reduced NAS, especially lobular inflammation and hepatocyte ballooning, liver triglyceride and blood glucose. This was associated with suppression of Protein Kinase C. (PKC)-ε, Glucose-6-phosphatase (G6pc) and Fructose 1, 6-biphosphatase (FBPase) in the liver. At week 12 (Fibrosis phase), solithromycin significantly reduced the area of fibrosis and showed reductions of NAS, blood glucose, G6pc and FBPase in a dose dependent manner. At week 20 (HCC phase), solithromycin macroscopically reduced the number, incidence and size of HCC. Solithromycin repressed NASH and fibrosis, and subsequently inhibited the development of HCC. Reductions in blood glucose, G6pc and FBPase suggest that suppression of gluconeogenesis pathway in NASH phase is an unexpected mechanism of solithromycin and could open a new avenue for the treatment of NASH-HCC.

Solithromycin is a novel macrolide antibiotic that has demonstrated strong immunomodulatory properties in vitro and in the cigarette smoke exposed mouse Chronic Obstructive Pulmonary Disease (COPD) model (Supplementary Figure 1) [12,13]. This off-target activity is well known as a class effect for macrolide antibiotics and has provided clinical benefit that enhances their antibacterial properties [14]. The antiinflammatory properties of macrolides have led to their clinical use in COPD and cystic fibrosis for chronic, long-term treatment [15,16]. Solithromycin has been tested in two large Phase 3 trials for the treatment of Community-Acquired Bacterial Pneumonia (CABP) and is being developed for other infections in adult and pediatric populations [17-19]. The data from these clinical trials in treating bacterial infections, along with a study in hepatic insufficiency patients and 90-day toxicology studies conducted in two animal species provide a rationale for the use of solithromycin over a longer treatment duration [20].

Keywords: NASH Treatment, Solithromycin, Gluconeogenesis, Diabetic NASH

Introduction

Non-Alcoholic Fatty Liver Disease (NAFLD) is highly prevalent. Population based studies suggest that as many as one in four persons worldwide may be affected [1]. This may even be higher in at-risk US populations [2]. NAFLD encompasses a spectrum of pathology ranging from simple bland steatosis to associated inflammation with or without fibrosis known as Non-Alcoholic Steatohepatitis (NASH) [3]. NASH is a risk factor for cirrhosis, which occurs in about 12-20% of affected individuals and represents the extreme end of the spectrum, associated with increased morbidity and mortality [4,5]. Early studies have reproducibly shown an association between NASH and insulin resistance, which led to multiple randomized, placebo-controlled studies of insulin sensitizers, most prominently PPAR-gamma agonists [6,7]. Other early interventions focused on antioxidants such as Vitamin E and the methyl donor betaine, which seem to play an important role in the transition from simple steatosis to steatohepatitis [7,8]. Unfortunately, these studies have failed to produce a safe and effective treatment for NASH due to relatively small effect size, rapid worsening and return to baseline when treatment is stopped and potential safety concerns. Some of the challenges with drug development have stemmed from the absence of animal models that closely reproduce human NASH without the need for drastic dietary, genetic or immunological manipulation [9]. As a result, research has shifted to new therapeutic options that focus on reducing inflammation, fibrosis and ideally, with both FXR agonists and dual PPAR-alpha and delta agonists currently being tested in advanced phase clinical trials [10,11]. Therefore, the need for safe, effective, orally administered therapies for NASH represents perhaps the greatest unmet need in liver disease today.

Solithromycin's anti-inflammatory/immunomodulatory properties are being explored in a Phase 2 COPD trial. Solithromycin achieves high concentrations in the respiratory tract and in the liver, the primary organ responsible for its metabolism and excretion. Solithromycin is well absorbed when administered orally which could be useful for long-term treatment and also lacks significant activity against anaerobic, Gram-negative intestinal microflora [21]. Hepatic inflammation plays a major role in the pathogenesis of NASH, therefore preventing such inflammation could be beneficial in stopping the progression of NASH [22]. Therefore, we explored the potential therapeutic benefit of solithromycin in a diabetic mouse model for NASH given its immunomodulatory properties, safety and tolerability, oral bioavailability and high liver concentrations. NASH occurs across a variety of human genetic backgrounds with obesity being only one contributing factor [23]. Diabetes is an established risk factor for NASH [24]. Although several rodent models are available to study NASH, we used the diabetic mouse model because the NASH phenotype is reproducible in diabetic C57BL/6J mice when fed a high fat diet and follows a predictable path from steatosis to inflammation and fibrosis to Hepatocellular Carcinoma (HCC) [25-28].

Methods

Mouse model

The diabetic mouse model of NASH in C57BL/6J mice fed a high fat diet is reproducible and follows a predictable progression of steatosis, hepatic inflammation, hepatocellular ballooning degeneration, fibrosis and HCC (Figure 1) [28].

Induction of NASH in mice

Mice were housed and cared for in accordance with the Japanese Pharmacological Society Guidelines for Animal Use. NASH was induced in C57BL/6J male mice by a single subcutaneous injection of 200 μg streptozotocin solution (STZ, Sigma-Aldrich, USA) two days after birth and feeding with a high fat diet, (HFD, 57 kcal% fat, cat#: HFD32, CLEA Japan, Inc., Japan) starting at 4 weeks of age and continuing for the duration of the experiments.

Treatment schedule

Solithromycin was suspended in 0.5% w/v methylcellulose + 0.2% Tween 80 and administered orally at doses of 5 mg/kg once daily,10 mg/kg twice daily, 25 mg/kg twice daily, 50 mg/ kg once daily and 100 mg/kg once daily. Groups of NASH mice were treated between (i) 4-8 weeks of age to examine the effect on early disease progression or (ii) 8-12 weeks of age to examine the effect of treatment after fibrosis had developed (100 mg/kg QD only), or (iii) 4 to 20 weeks of age (100 mg/kg QD only), to examine the effect on hepatocellular carcinoma development.

Macroscopic analyses of liver

Livers were examined for morphology, fat streaks, and gross, swollen tumor nodules. The diameter of tumor nodules was measured with a caliper and those with a diameter greater than 2 mm were counted.

Measurement of whole blood, plasma, serum and liver biochemistry

Non-fasting blood glucose was measured in whole blood. Plasma insulin levels were quantified by the ultra-sensitive Mouse Insulin ELISA kit (Morinaga Institute Biological Science, Inc., Japan). Plasma Macrophage Migration Inhibitory Factor (MIF) was measured using MIF ELISA kit (Kamiya Biomedical, USA) and IL-22 was measured using Quantikine ELISA Mouse/Rat IL-22 Immunoassay kit (R&D Systems, USA). For serum biochemistry, blood was collected without anticoagulant. Serum triglyceride, HDL-, LDL-, VLDL-cholesterol and chylomicron concentrations were quantified by HPLC at Skylight Biotech Inc. (Japan). Liver total lipid-extracts were obtained by Folch's method [29]. Liver triglyceride levels were measured by Triglyceride E-test (Wako Pure Chemical Industries, Japan). Liver diacylglycerol levels were measured by Thin Layer Chromatography (TLC) at Toray Research Center (Japan).

Histological analyses

NAFLD Activity Score (NAS) was determined on H&E stained sections as previously described (Supplementary Table 1) [30]. Collagen was stained using picro-Sirius red solution (Waldeck, Germany). Frozen liver sections were used for immunohistochemistry, using anti-F4/80 antibody (BMA Biomedicals, Switzerland) to determine macrophages or a 200- fold dilution of anti-Gr-1 antibody (Abcam, USA) to determine neutrophils. For quantitative analysis of fibrosis and inflammation areas, bright field images of prepared sections were captured around the central vein using a digital camera (DFC280; Leica, Germany) at 200-fold magnification and positive areas were measured using Image J software (National Institute of Health, USA). Gr-1 positive cells in 5 fields/section were counted. Gene expression in liver -Quantitative RT-PCR Total RNA was extracted from liver samples. One μg of RNA was reverse-transcribed using a reaction mixture and real-time PCR was performed. The relative mRNA expression level was calculated using the expression of each gene normalized to that of the reference gene 36B4, a housekeeping gene coding for an acidic ribosomal protein [31]. The mRNA expression of a variety of genes involved in glucose and lipid metabolism, inflammatory cytokines and collagen deposition and fibrosis were measured. Farnesoid X Nuclear Receptor (FXR) assays Reporter cells expressing hybrid FXR comprising the N-terminal Gal4 DNA binding domain fused to the ligand binding domain of the human FXR were used to determine agonistic and antagonist solithromycin activity. The plates were incubated for 24 hours at 37oC prior to analysis with a luciferase detection reagent. GW4064, a known agonist of the FXR receptor, was used as the control. To determine antagonistic activity, the cells were mixed with 2X EC80 concentration of GW4064 and the decrease in response to GW4064 was measured.

Statistical analyses

Statistical analyses were performed using Bonferroni Multiple Comparison Test or Student t-test on GraphPad Prism 4 (GraphPad Software, USA). P values < 0.05 were considered statistically significant. Results were expressed as mean +/- SD. 

Results

Liver weight and liver-to-body weight ratio percent 

Liver weight and liver-to-body weight data are presented in Supplementary Table 2. The liver-to-body weight ratio reduction also demonstrated a graded dose response but with no statistically significant decrease (p >0.05) at the 10 mg/kg BID or the 5 mg/kg QD groups. The 25 mg/kg BID treatment with solithromycin from 4-8 weeks of age resulted in a significant decrease (P<0.001) in mean liver weight compared with the Vehicle group (Vehicle: 1411 +/- 173 mg, Solithromycin: 1087 +/- 97 mg). The liver-to-body weight ratio was significantly reduced (p<0.01) in the 25 mg/kg BID group but was not further decreased in the 50 mg/kg QD or the 100 mg/kg QD dose , with maximum effect being noted at the 25 mg/kg BID dose.

Histological analyses and NAFLD activity scores 

At 8 weeks of age, liver sections from the Vehicle group exhibited severe micro- and macrovesicular fat deposition with predominantly macrovesicular fat in the NASH mice, hepatocellular ballooning and inflammatory cell infiltration. Consistent with these observations, NAS significantly increased (p<0.001) in the Vehicle group (4.9 +/- 0.8) compared to the Normal group (0.0 +/- 0.0). A clear dose-response in histological improvement of multiple histologic parameters was observed with the maximum effect noted at the 50 mg/kg QD dose (Figure 2 A-D). At the 50 mg/kg QD dose, liver sections from the Solithromycin group showed marked improvements in hepatocellular ballooning and inflammatory cell infiltration, with significant reduction (p<0.0001) in NAS compared with the Vehicle group (Vehicle: 5.4 +/- 0.5, Solithromycin: 3.0 +/- 0.9) (Figure 2A). Individual components of the NAFLD activity score, which include lobular inflammation, hepatocellular ballooning degeneration and teatosis are shown in Supplementary Table 2. Representative photomicrographs of the H&E stained liver sections are shown in (Figure 2B and 2C). There was no further increase in histological improvement at the 100 mg/kg QD dose of Solithromycin in comparison with the 50 mg/kg QD dose. Solithromycin 10 mg/kg BID and 25 mg/kg BID treated groups also showed marked improvements in hepatocellular ballooning and inflammatory cell infiltration, with significant reduction (p<0.001 at the 25 mg/kg BID dose) in NAS compared to the Vehicle group (Figure 2D). There was no significant difference (p>0.05) in NAS between the Vehicle group and the Solithromycin 5 mg/kg QD treated group (Supplementary Table 3). 

Macrophages in liver sections- F4/80 immunohistochemistry 

Liver sections from the Vehicle group showed an increase in the number of macrophages and inflammation area size as measured by F4/80-positive cells in the liver lobule compared to the Normal group. The percentage of F4/80-positive areas significantly increased in the Vehicle group compared to the Normal group. There were no statistically significant differences (p>0.05) in the area of F4/80-positive macrophage infiltration between the Vehicle group and any of the Solithromycin dose groups. 

Neutrophils in liver sections

Gr-1 immunohistochemistry on liver sections from the Vehicle group showed increased infiltration of neutrophils (Gr-1- positive cells) in the liver lobule compared to the Normal group. In NASH mice treated between 4-8 weeks with 5 mg/kg QD, 10 mg/kg BID and 25 mg/kg BID the decrease in the number of Gr-1 positive inflammatory cells was not statistically significant (p>0.05). Gr-1 immunohistochemistry was not evaluated in the other dose groups. However, inflammation was evaluated in the H&E sections in all dose groups. Lobular inflammation rating in all the dose groups indicates a decrease in the inflammatory score with increasing dose with a similar maximum effect noted at the 25 mg/kg BID and the 50 mg/kg QD dose groups (Supplementary Table 3). No further decrease was noted with the 100 mg/kg QD treatment group. 

CK-18 in liver sections

CK-18 is a major intermediate filament protein in the liver. Increased CK-18 staining reflects hepatocellular damage. Immunohistochemistry of liver sections from the Vehicle group showed a strong intensity of immunostaining for CK-18 in the degenerative hepatocytes compared to the Normal group. There were no obvious changes in CK-18 immunostaining between the Vehicle group and in the 5 mg/kg QD, 10 mg/kg BID and the 25 mg/kg BID dose groups of Solithromycin. CK-18 was not evaluated in the other dose groups or in other repeated experiments as it was negative in this experiment.

Collagen deposition and measurement of fibrosis

Picro-sirius red staining of liver sections from the Vehicle group showed increased collagen deposition in the pericentralregion of the liver lobule compared to the Normal group. The percentage of fibrosis area (Sirius red-positive area) significantly increased in the Vehicle group compared to the Normal group (p<0.001). Compared to the Vehicle group, the fibrosis area tended to decrease with increasing solithromycin dose. The Solithromycin 25 mg/kg BID dose group had a significant decrease in fibrosis (p<0.05) (Supplementary Table 4). NASH mice in the diabetic mouse model at 4-8 weeks had not always progressed to obvious fibrosis in the Vehicle treated group and it was therefore difficult to evaluate the effect of solithromycin consistently at this early time-point. Therefore, the effect of solithromycin treatmenton fibrosis was evaluated in NASH mice treated between 8-12 weeks, beginning treatment at 8 weeks, at which time fibrosis have progressed in the vehicle group compared to the normal group. The fibrois area in the Solithromycin treated group (100 mg/kg QD) was significantly decreased (p<0.01) compared to the vehicle group at week 12 (Figure 3, Supplementary Table 4). Lower doses were not tested in the 8-12 week treatment experiments.

mRNA expression of inflammatory and collagen/fibrosis markers

Relative mRNA expression in the liver of inflammatory markers such as TNF-α, MCP-1, MMP-9 was unaffected by solithromycin (Supplementary Table 5). Among the collagen and fibrosis markers tested, there were no significant differences (p>0.05) in the relative mRNA expression of collagen Type 1, Alpha-SMA, TIMP-1, and TGF-β (Supplementary Table 5).

Serum Inflammatory Markers

Plasma MIF levels were not changed in NASH mice and were the same between the Vehicle group and the Normal group. MIF levels in the Solithromycin 25 mg/kg group were significantly increased (p<0.01) compared to the Vehicle group. There were no significant differences (p>0.05) in plasma MIF levels between the Vehicle group and the other Solithromycin groups. Plasma IL-22 levels in the Vehicle group were significantly decreased (p<0.01) compared to the Normal group. There were no differences in plasma IL-22 levels between the Vehicle group and any of the Solithromycin treated groups.

Plasma Alanine Aminotransferase (ALT)

Plasma ALT levels in the Vehicle group tended to increase compared to the Normal group. There were no significant differences (p>0.05) in plasma ALT levels between the Vehicle group and any of the Solithromycin treated groups. AST levels were not measured in any of the experiments, as no changes were observed with ALTs and the AST changes in humans have been lower than that of ALTs [17].

Whole blood glucose lowering effect

The vehicle group showed a significant increase (p<0.001) in whole blood glucose levels compared to the Normal group. In NASH mice, whole blood glucose was reduced in the Solithromycin treated groups compared to the vehicle group. A dose-response was noted with the effect being significant at the 25 mg/kg BID and 50 mg/kg QD doses (p<0.05 and p<0.01, respectively) (Figure 4A and 4B, Supplementary Table 6). The 50 mg/kg QD dose gave the maximal response (Vehicle: 628 +/- 85 mg/dL, Solithromycin: 380 +/- 170 mg/dL) (Figure 4A). There were no significant differences (p >0.05) in whole blood glucose levels between the Vehicle and either the Solithromycin 5 mg/kg QD or 10 mg/kg BID treatment groups. As expected, plasma insulin levels in the vehicle and Solithromycin groups were low and not significantly different (p>0.05) in any of the treatment groups with the single exception of recovery noted in the 100 mg/kg QD group treated from 4-8 weeks (Supplementary Table 6). Unlike the group treated from 4-8 week at the same dose, the 100 mg/ kg QD 8-12 week treatment group did not show increased insulin, but did show a statistically significant plasma glucose lowering effect (p<0.01) (Figure 4C and Supplementary Table 6). 

Glucose-6-phosphatase (G6pc) and Fructose-1,6-bisphosphatase (FBPase) relative mRNA expression

G6pc and FBPase mRNA expression levels were significantly up-regulated (p<0.001) in the Vehicle group compared to the Normal group. G6pc and FBPase relative mRNA expression levels were significantly down-regulated in all Solithromycin dose groups, including the 5 mg/kg QD dose group (p<0.01 for G6pc and p<0.05 for FBPase) when compared to the Vehicle group (Figure 5 A-D and Supplementary Table 6). A dose-response was noted with both G6pc and FBPase relative mRNA expression. 


Glucokinase (Gck), Phosphoenolpyruvate Carboxykinase 1 (Pck1), Protein Kinase C epsilon (PKCε), and Phosphatidylinositide 3-Kinase (PI3K) mRNA Expression

Gck mRNA expression levels were significantly downregulated (p<0.01) in the Vehicle group compared to the Normal group. There were no significant differences (p>0.05) in Gck mRNA expression levels between the Vehicle group and any doses of the Solithromycin groups. Pck1 mRNA expression was significantly up-regulated in the Vehicle group compared to the Normal group (p<0.05). There were no significant differences (p>0.05) in Pck1 mRNA expression levels between the Vehicle group and any doses of the Solithromycin groups. PKCε mRNA expression was down-regulated in the Solithromycin treatment group and was tested only in the 100 mg/kg QD 4-8 (Solithromycin 1.13 + 0.26 vs Vehicle 1.65 + 0.45) and 8-12 week (0.89 + 0.44 vs. 1.25 + 0.46) groups. The down-regulation reached statistical significance (p<0.05) only in the 4-8 week group although a decrease was also observed in the 8-12 week group. PI3K mRNA expression levels were significantly up-regulated (p<0.05 ) in the 100 mg/ kg QD 4-8 week Solithromycin group (2.29 + 0.56 vs. 1.65 + 0.21) compared to the Vehicle group. However, in the 8-12 week Solithromycin group, the observed increase was not statistically significant (1.89 + 0.34 vs. 1.64 + 0.51, p>0.05).

GLUT-2 mRNA expression

There were no differences in Glut 2 mRNA expression levels between the Vehicle group, the Normal group and the Solithromycin treated groups.

Serum and liver lipids

At 8 weeks of age, serum total-cholesterol levels in the Vehicle group were increased compared to the Normal group. There were no significant differences in serum total-cholesterol levels between the Vehicle group and any doses of the Solithromycin groups (p>0.05). LDL-cholesterol was significantly increased (p<0.001) in the 25 mg/kg BID and 100 mg/kg QD 8-12 week treatment groups (Table 1). The detailed lipid analysis was not conducted in the first experiment that tested 50 mg/kg QD. Lower doses did not reach statistical significance. In contrast, serum chylomicrons, VLDL and serum triglycerides were all increased in the Vehicle treated group, but were significantly decreased in the 25 mg/kg BID group (p< 0.05, p< 0.05, and p< 0.01, respectively). This trend was not observed in the lower Solithromycin dose groups. Also, serum HDL-cholesterol levels showed a dose responsive tendency to increase in the 5 mg/kg QD, 10 mg/kg BID, and 25 mg/kg BID Solithromycin treated groups, however statistical significance was not achieved.

Liver triglyceride and diacylglycerol levels were increased in the Vehicle group. In the 100 mg/kg QD 4-8 week group, liver triglycerides were decreased (p<0.001). Statistical significance was not achieved in the 5 mg/kg QD, 10 mg/kg BID, and 25 mg/kg BID Solithromycin groups, but a tendency to decrease liver triglycerides was noted in the 25 mg/kg BID group. Liver diacylglycerol showed a tendency to decrease in the 4-8 week 100 mg/kg QD group, but did not reach statistical significance in any Solithromycin group tested (Data not shown). 

Effect on HCC development: Macroscopic analyses of liver at week 20

All Vehicle treated NASH mice had hepatic tumor nodules at 20 weeks. The Solithromycin group showed a significant decrease (p<0.01) in the number of visible tumor nodules formed on the liver surface compared to the Vehicle group (Figure 6A). In addition, the Solithromycin group showed a significant decrease (p<0.01) in the maximum diameter of visible tumor nodules in comparison to the Vehicle group (Figure 6B).

Activation of FXR

Solithromycin did not show agonistic activity in the human FXR assays (Supplementary Figure 2). Furthermore, at noncytotoxic concentrations, solithromycin was not antagonistic to FXR.

Discussion

In the diabetic mouse model of NASH, solithromycinadministration resulted in hepatic histological improvement as evidenced by reduction in NAFLD activity scores that reflect a decrease in inflammation and hepatocyte ballooning degeneration. In addition, hepatic tissue and inflammation improved. Many of the solithromycin treated groups showed a reduction in fibrosis as well. Lastly, both the number and diameter of hepatocellular carcinoma nodules detected at 20 weeks were reduced in the Solithromycin group compared to Vehicle treated group. The experiments were designed to explore markers of inflammation, such as cytokines, glucose and lipid metabolism, cell proliferation, collagen deposition, FXR ligand binding and other factors associated with diabetes and NASH. The exact mediators of the anti-inflammatory effect remains unclear, as most of the inflammatory markers examined were not consistently decreased. The effect on the down regulation of enzymes involved with gluconeogenesis, even at the lowest dose of 5 mg/kg QD tested, as well as the lowering of blood glucose in the solithromycin treated mice were notable and may shed light on the mechanism of action of this macrolide for NASH. Suppression of gluconeogenesis appears to be an important contributing mechanism of action for solithromycin in improving NASH. A marked increase in gluconeogenesis is purported to be an underlying component of NASH in human subjects [32]. The enzymes FBPase and G6pc involved in the gluconeogenic pathway are regulated by PKCε and have been described as potential targets for treating diabetes [33]. These enzymes play a role in the late stages of gluconeogenesis while earlier enzymes in the pathway, such as Gck and Pck 1 are unaffected. Other work has suggested a link between elevated TCA cycle function and both hepatic insulin resistance and fatty liver in a mouse model. In this model, impaired insulin signaling resulted in elevated in vivo gluconeogenesis [34]. In the current studies, solithromycin suppressed hepatic mRNA expression of two key gluconeogenic enzymes, FBPase and G6pc. This appears to be the mechanism of glucose lowering in the absence of changes in insulin in this model system, functionally reversing hepatic insulin resistance [23,35]. The important role of PKCε in regulating lipid-induced hepatic insulin resistance may be at play as its mRNA expression level was significantly reduced even when hepatic diacylglycerol content was unchanged. PKCε (phosphoenolpyruvate carboxykinase 1), can phosphorylate and subsequently activate AKT (Supplementary Figure 3). Activated AKT suppresses the expression of gluconeogenic enzymes such as G6pc and FBPase by the phosphorylation and exclusion of the forkhead protein FOXO1 [36]. In this study, the improvement in histological parameters of inflammation was also accompanied by the improvement in fibrosis among solithromycin treated groups. The link between hyperglycemia and progression of fibrosis has now been demonstrated in a number of studies with Connective Tissue Growth Factor (CTGF) among implicated intermediates [37].

Although LDL cholesterol was increased in some of the solithromycin treated groups, it may be balanced by a simultaneous increase in HDL and decrease in VLDL cholesterol and chylomicrons, with total cholesterol remaining unaffected.Liver triglycerides were significantly decreased at the highest dose tested (100 mg/kg QD of solithromycin) (p<0.001) while liver Diacylglycerol (DAG) was unaffected. The FXR pathway, which is a target for current NASH experimental treatments (10), was not impacted by solithromycin administration. 

We explored whether the potential therapeutic effect of solithromycin could be secondary to its antibacterial activity. Endotoxin released from Gram-negative intestinal bacteria has been linked with increased hepatic inflammation [38]. Solithromycin does not have activity against Gram-negative anaerobic bacteria even though it does affect aerobic Grampositive and Gram-negative intestinal microflora and can decrease aerobic enteric bacterial counts, such as Escherichia coli at the doses tested. In our studies with IL-22, a cytokine linked to gut flora, which is depressed in diabetic, obese mice [39], there was no significant change as a result of solithromycin administration. The 50 mg/kg dose of solithromycin that appeared to optimally improve hepatocellular ballooning degeneration in NASH, is equivalent to 200 mg at the human dose, half the dose used to treat CABP. It was also noted that the 25 mg/kg BID dosing was less effective in all responses evaluated compared to the 50 mg/ kg QD dosing. This indicates that the plasma Cmax of solithromycin may be more important that the plasma AUC for impact on the treatment of NASH. This is in contrast to dosing regimens used for treating bacterial infections where AUC is more critical than Cmax for macrolide antibiotics. Solithromycin is well absorbed after oral administration (65% at the 400 mg dose). Only a very small amount of the drug is found in the intestinal tract (<15% of unchanged solithromycin is found in the feces (Unpublished data, Cempra, Inc). Solithromycin produced statistically significant reductions in hepatocellular ballooning degeneration, fibrosis, and HCC at a daily dose that is equivalent or lower than the Human Equivalent Dose (HED). Furthermore, it has been well tolerated and effective in two global Phase 3 trials to treat moderate to moderately severe CABP.

The observed decrease in progression to HCC in the diabetic mouse model of NASH is a novel and exciting finding. Solithromycin is not known to directly suppress pathways related to tumorigenesis and tumor growth, therefore, its effects are most likely attributed to the regulation of glucose, lipids and inflammation. Indeed, recent data suggest that control of hyperglycemia in a high fat diet mouse model of NASH may reduce the development of hepatic tumorigenesis [40].

Although solithromycin is an antibiotic under clinical development for the treatment of bacterial infections, like older macrolides, such as azithromycin, it has additional properties. Macrolide antibiotics have been historically known to have offtarget activities and are used clinically [15,16,41] and for long-term treatment for diseases such as COPD and cystic fibrosis. In this study, we have demonstrated that solithromycin was effective in significantly reducing inflammation, fibrosis, and NAFLD scores in a diabetic and high-fat diet-induced mouse model of NASH. The availability of a large safety database together with the results in this mouse model support the exploratory development of solithromycin for the treatment of NAFLD and NASH in humans. Solithromycin was effective in reducing blood glucose, without significant improvement in insulin levels in the NASH mice. The "anti-NASH" activity is proposed to be through solithromycin's immunomodulatory properties and through the suppression of gluconeogenesis. An exploratory trial in patients with biopsy-proven NASH is currently underway (ClinicalTrials. gov: NCT02510599).

Acknowledgements

We thank Dr. Kara Keedy and Evan Martens for helping with editing the manuscript. Grant details/support not applicable. Disclosures/Potential Conflict of Interest Prabhavathi Fernandes and David Oldach are employees of Cempra Inc., receive a salary, and hold Cempra shares. Taishi Hashiguchi, Yuka Shirakata and Hiroyuki Yoneyama work for the Stelic Institute & Co. Inc, Tokyo, and conducted the work under a paid contract with Cempra, Inc. Pierre M. Gholam is a consultant to Cempra Inc. and is currently the Clinical Investigator studying solithromycin in a Phase 2 clinical trial at the Liver Center of Excellence, University Hospitals Case Medical Center, Cleveland, OH.

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Figure 1: Progression of NASH to development of HCC in the diabetic mouse model of NASH.


Figure 2: Histological changes related to administration for solithromycin from 4 to 8 weeks of age in the diabetic mouse model for NASH; (A) NAFLD activity score in the 50 mg/kg QD Solithromycin group; (B) Components of the NAFLD Activity Score in the 50 mg/kg QD group; (C) H&E stained section of liver showing reduction in hepatocyte ballooning degeneration (50 mg/kg QD from 4-8 weeks) (i) Vehicle control (ii) Solithromycin treated (50 mg/kg QD from 4-8 weeks); (D) NAFLD activity score dose response in the 5 mg/kg QD, 10 mg/kg BID and the 25 mg/kg BID treatment groups.

Figure 3: Reduction of fibrosis in NASH mice at 12 weeks when treated from 8-12 weeks with Solithromycin 100 mg/kg QD.

Figure 4: Effect of Solithromycin treatment on plasma blood glucose levels; (A) 50 mg/kg QD; (B) Dose response noted with administration of 5 mg/kg QD, 10 mg/kg BID and 25 mg/kg BID from 4-8 weeks; (C) 100 mg/kg QD from 8-12 weeks.

Figure 5: Effect of Solithromycin on Glucose-6–phosphatase (G6pc) and Fructose-1,6- bisphosphatase (FBPase) relative mRNA expression; (A) Dose response noted for G6pc in 5 mg/kg QD, 10 mg/kg BID, and 25 mg/ kg BID treatment groups; (B) Dose response noted for FBPase in 5 mg/ kg QD, 10 mg/kg BID and 25 mg/kg BID treatment groups; (C) G6pc mRNA suppression with Solithromycin 100 mg/kg QD; (D) FBPase mRNA suppression with Solithromycin 100 mg/kg QD.


Figure 6: The effect of Solithromycin (100 mg/kg QD, 4-20 weeks) on (A) the number of visible tumor nodules, and (B) the size of visible tumor nodules at 20 weeks.


Table 1: Effect of Solithromycin treatment (from 4-8 weeks of age) on serum lipids in NASH diabetic mice fed a high fat diet.


Supplementary Tables and Figures:

Supplementary Table 1: Definition of NASH components for calculation of NAFLD Activity scores.

Supplementary Table 2: Effect of Solithromycin on Liver Weights and Liver-to-Body Weight Ratio

Supplementary Table 3: NAFLD Activity Scores calculated from scoring H&E liver sections for steatosis, lobular inflammation and hepatocyte ballooning degeneration

Supplementary Table 4: Effect of Solithromycin on fibrosis seen in H&E sections of NASH mice

Supplementary Table 5: Effect of Solithromycin on relative mRNA expression of inflammatory and collagen/fibrosis markers in the liver of NASH mice

Supplemental Table 6: Effect of Solithromycin treatment on plasma insulin, blood glucose and relative mRNA expression of glucose-6-phosphatase (G6pc) and fructose-1,6,-bisphosphatase (FBPase) in NASH mice

Supplementary Figure 1: Chemical structure of solithromycin

Supplementary Figure 2: Effect of Solithromycin in the Human FXR agonism assay
Supplementary Figure 3: Proposed Mechanism by which Solithromycin Blocks Gluconeogenesis (G6pc and FBPase)

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