Inhibition of IKKɛ and TBK1 Improves Glucose Control in a Subset of Patients with Type 2 Diabetes

Abstract

Numerous studies indicate an inflammatory link between obesity and type 2 diabetes. The inflammatory kinases IKKɛ and TBK1 are elevated in obesity; their inhibition in obese mice reduces weight, insulin resistance, fatty liver and inflammation. Here we studied amlexanox, an inhibitor of IKKɛ and TBK1, in a proof-of-concept randomized, double-blind, placebo-controlled study of 42 obese patients with type 2 diabetes and nonalcoholic fatty liver disease. Treatment of patients with amlexanox produced a statistically significant reduction in Hemoglobin A1c and fructosamine. Interestingly, a subset of drug responders also exhibited improvements in insulin sensitivity and hepatic steatosis. This subgroup was characterized by a distinct inflammatory gene expression signature from biopsied subcutaneous fat at baseline. They also exhibited a unique pattern of gene expression changes in response to amlexanox, consistent with increased energy expenditure. Together, these data suggest that dual-specificity inhibitors of IKKɛ and TBK1 may be effective therapies for metabolic disease in an identifiable subset of patients.

Keywords: amlexanox; clinical trial; energy expenditure; fatty liver; gene expression; inflammation; obesity; protein kinase.

Figure 1. Study design and insulin sensitivity measurements from the open-label study

a) Summary of the study protocols. † indicates protocol elements that only apply to the open-label study. b) Baseline characteristics of the six open-label study participants. Metabolic data of the six patients included in the study at baseline and 12 weeks are shown: c) Hemoglobin A1c % (HbA1c). d) Fasting glucose. e) Fasting insulin. (f) Insulin resistance calculated by the homeostatic model assessment linear estimation, HOMA-IR. g–i) Hyperinsulinemic euglycemic clamp: g) Rate of glucose disappearance (Glucose Rd). h) body weight-normalized glucose Rd and (i) Hepatic glucose production at fasting state. j) Percent fat in liver, as determined via MRI using Dixon method. k) Glucose (left panel) insulin (middle panel) and FFAs (right panel) during the mixed meal test. Data presented as individual values (c–h) or mean ± s.e.m. (k). n = 6, except in g and h, as the 6^th patient did not achieve steady state during the clamp performed at 12 weeks, where n = 5. * indicates p-value ≤ 0.05, # indicates p-value ≤ 0.10 (two-tailed paired t-test). DEXA, Dual Emission X-ray Absorptiometry; t.i.d., ter in die, three times a day. See also Figure S1, and Table S1.

Figure 2. Weight loss and gene expression changes in the open label study subjects

a) Body weight (left panel) and body weight represented as percentage of baseline body weight (right panel) during 12-week treatment and 4-week washout observation period. b) Relative expression of UCP family of genes encoding mitochondrial inner membrane protein. c) Correlation of relative UCP1 expression to weight loss after 12 weeks of treatment. d) Relative expression of ADRB family of genes encoding β-adrenergic receptors. Relative expression of beige fat markers: (e) FGF21, (f) DIO2, (g) PPARGC1A, PPARGC1B, PRDM16, and ELOVL3. f) Correlation of DIO2 expression and weight loss (bottom panel). h) Relative expression of IL4. i) Relative expression of ADIPOQ, the gene encoding adiponectin. n = 6. ** indicates p-value ≤ 0.01; * indicates p-value ≤ 0.05; # indicates P-value ≤ 0.10 (two-tailed paired t-test). c, f Pearson’s correlation with 95% CI. AU, arbitrary unit.

Figure 3. Measures of glycemic control in the double blind, placebo-controlled trial

a) Consort diagram: Schematic of randomized double blind placebo-controlled trial. For details on evaluable n of secondary and exploratory end point analyses, refer to Table S2. Difference from baseline in b) Hemoglobin A1c and (c) fructosamine in the placebo- and amlexanox-treated patients left panels; right panels display baseline and 12-week values for each patient connected with a line. d) Insulin and (e) glucose during the mixed meal test. f) Insulin ratio (week 12 divided by baseline area under the curve for insulin measured during the mixed meal test). b: n = 18 placebo, n = 20 amlexanox; c–f: n = 17 placebo, n = 20 amlexanox (n = 7 A–R). ** indicates p-value ≤ 0.01 and * indicates p-value ≤ 0.05 (two tailed t-test, b, c, e unpaired, d paired). Data presented as individual data and mean ± s.e.m. (b, c, f) or mean ± s.e.m. only (d, e). See also Figure S2–S4, and Table S3 and S4.

Figure 4. Baseline differences in responders versus non-responders

Serum levels of (a) amlexanox and (b) one of its metabolites at baseline and 12 weeks. a, b) n = 18 placebo, 7 amlex-R (Amlexanox-treated, responder) and 13 amlex-NR (amlexanox treated, non-responder). See also Table S5. c) Genes whose expression was significantly different in responders versus non-responders at baseline in RNA-sequencing analysis. Values presented as log (fold change) in responders relative to non-responders; (n = 4 per group). d) Baseline CRP values in the placebo-controlled trial in the placebo- and drug-treated patients. The latter group is further divided into responders and non-responders. Baseline CRP values for one of the placebo treated patients was excluded as it was obviously compromised. n = 17 placebo, 7 amlex-R, 13 amlex-NR. e) Correlation of baseline CRP values with change in HbA1c from baseline to 12 weeks in amlexanox-treated patients (left panel, n = 20) and placebo controls (right panel, n = 17). Note the significant inverse relationship in the drug treated group compared to lack of correlation in the placebo-treated group. f) Correlation between baseline BMI and serum CRP at baseline in all patients (left panel; p = 0.031, Pearson’s r = 0.36, n = 37,) and FMI (fat mass index) vs. serum CRP in all patients (right panel) at baseline (p =0.0003, Pearson’s r = 0.56, n=37). g) Correlation between liver fat and serum CRP at baseline in patients who were evaluated for liver fat (p = 0.035, Pearson’s r = 0.49, n = 19). # indicates p-value ≤ 0.10, * indicates p-value ≤ 0.05 (two tailed t-test). Data presented as individual data and mean ± s.e.m or 95% CI. CRP: C-reactive protein. BMI: body mass index. See also Figure S4.

Figure 5. Responder specific changes in serum IL-6 and subcutaneous fat gene expression

a) Serum IL-6 levels over the course of the trial: 12 weeks of treatment and 4 weeks of observation period post treatment. b–f, i) Relative expression of genes in the subcutaneous fat at baseline and after 12 weeks of treatment. b–e) Relative expression of beige fat associated genes: (b) UCP1 (Ct values in the non-responder (baseline: 35.1, 12-week: 35.9) and placebo (baseline: 34.7 12-week: 34.4) were not changed after treatment, while the Ct value in the responder was reduced (baseline: 35.9 12-week: 33.8). While these high Ct values indicate low abundance of UCP1, good linearity was observed in the standard curve from Ct 30 to Ct 35), (c) DIO2, (d) PRDM16 (left panel), FGF21 (right panel), and (e) COX5B. f) Relative expression of CCL2. g) Heat map illustrating the genes that were significantly changed from baseline to 12 weeks in a paired analysis of the RNA-sequencing data within each of the three groups. h) Heat map illustrating genes from different pathways that were differentially regulated by treatment in the responders (*HSPB1 was also significantly upregulated in the non-responder group). i) Relative expression in the subcutaneous fat at baseline and after 12 weeks of treatment of LIPE (top panel) and FASN (bottom panel). See also Table S6 and S7. (a: n = 17 placebo, 7 amlex-R and 13 amlex-NR; c–f, i: n = 15 placebo, 7 amlex-R and 13 amlex-NR; g,h: n = 4 per group * indicates p-value ≤ 0.05; # indicates P-value ≤ 0.10 (two-tailed t-test a,unpaired, b–i, paired). Data presented as mean ± s.e.m. Amlex-R: amlexanox-treated responder, Amlex-NR: amlexanox-treated, non-responder.