OBJECTIVE

Type 2 diabetes pathophysiology is characterized by dysregulated glucagon secretion. LY2409021, a potent, selective small-molecule glucagon receptor antagonist that lowers glucose was evaluated for efficacy and safety in patients with type 2 diabetes.

RESEARCH DESIGN AND METHODS

The efficacy (HbA1c and glucose) and safety (serum aminotransferase) of once-daily oral administration of LY2409021 was assessed in two double-blind studies. Phase 2a study patients were randomized to 10, 30, or 60 mg of LY2409021 or placebo for 12 weeks. Phase 2b study patients were randomized to 2.5, 10, or 20 mg LY2409021 or placebo for 24 weeks.

RESULTS

LY2409021 produced reductions in HbA1c that were significantly different from placebo over both 12 and 24 weeks. After 12 weeks, least squares (LS) mean change from baseline in HbA1c was –0.83% (10 mg), –0.65% (30 mg), and –0.66% (60 mg) (all P < 0.05) vs. placebo, 0.11%. After 24 weeks, LS mean change from baseline in HbA1c was –0.45% (2.5 mg), –0.78% (10 mg, P < 0.05), –0.92% (20 mg, P < 0.05), and –0.15% with placebo. Increases in serum aminotransferase, fasting glucagon, and total fasting glucagon-like peptide-1 (GLP-1) were observed; levels returned to baseline after drug washout. Fasting glucose was also lowered with LY2409021 at doses associated with only modest increases in aminotransferases (mean increase in alanine aminotransferase [ALT] ≤10 units/L). The incidence of hypoglycemia in the LY2409021 groups was not statistically different from placebo.

CONCLUSIONS

In patients with type 2 diabetes, glucagon receptor antagonist treatment significantly lowered HbA1c and glucose levels with good overall tolerability and a low risk for hypoglycemia. Modest, reversible increases in serum aminotransferases were observed.

Type 2 diabetes is characterized by chronic hyperglycemia as a result of impaired insulin secretion and action. Dysregulated glucagon secretion (13) and resultant hepatic glucose overproduction are also characteristic pathophysiological features and contribute to hyperglycemia in type 2 diabetes (410). Previous studies with LY2409021 have demonstrated that glucagon receptor antagonism inhibits glucose production in response to exogenous hyperglucagonemia in healthy volunteers and improves hyperglycemia in patients with type 2 diabetes through reductions in both fasting and postprandial glucose (11,12). A 28-day phase 1 study of LY2409021 in patients with type 2 diabetes demonstrated improvement in glycemic parameters at doses of 5–90 mg administered once daily (12). However, reversible, dose-related increases in the levels of serum aminotransferases were observed in that study (12). Similar reversible increases in serum aminotransferases have been noted with other glucagon receptor antagonists, including a human glucagon receptor monoclonal antibody (13), other small molecules (1417), and an antisense oligonucleotide targeting glucagon receptor expression (18).

In the present phase 2a and 2b studies, we further examined the glucose-lowering efficacy and safety of LY2409021 by comparing the relationships between doses of LY2409021 that lower HbA1c and glucose levels (efficacy) and those that increase serum aminotransferase levels (hepatic safety). The phase 2b study reported here represents the largest and longest clinical trial of a glucagon receptor antagonist reported to date.

Study Design

Both phase 2a (NCT00871572) and phase 2b (NCT01241448) studies were conducted in accordance with regulatory standards of good clinical practice, the Declaration of Helsinki, and all applicable local regulations. Study protocols were approved by each site’s ethical review board. All patients provided written informed consent before initiation of study procedures. The phase 2a study was a multicenter, randomized, double-blind, placebo-controlled, parallel-group study with a 1-week single-blind placebo lead-in period, a 12-week double-blind active treatment period, and a 4-week blinded posttreatment washout/follow-up period (Supplementary Fig. 1A). Patients were randomly assigned to one of the four treatment groups (Supplementary Fig. 1A) in a 1.5:2.1:1:1 ratio (60 mg:30 mg:10 mg:placebo). Unequal allocation was used to maximize the exposure to 30 mg LY2409021. Stratification was based on the absence or presence of preexisting metformin therapy and on study site. The phase 2b study was a multicenter, randomized, double-blind, placebo-controlled, parallel-group study consisting of a 1-week single-blind placebo lead-in period, a 24-week active treatment period, and a 4-week blinded posttreatment washout/follow-up period (Supplementary Fig. 1B). Patients were randomly assigned to one of the four treatment groups (Supplementary Fig. 1B) in a 1:1:1:1 ratio (20 mg:10 mg:2.5 mg:placebo). Stratification was based on preexisting metformin therapy, baseline HbA1c level (< or ≥8.2% [66 mmol/mol]), and baseline alanine aminotransferase (ALT) levels (≤ or > the upper limit of normal [ULN]). Patients were discontinued from study participation if they had fasting glucose levels >270 mg/dL on three or more separate days over any 2-week period between randomization and week 6 of treatment, fasting glucose levels >240 mg/dL on three or more separate days over any 2-week period between weeks 6 and 12, or fasting glucose levels >200 mg/dL or HbA1c levels >8.0% (64 mmol/mol) from week 12 to week 24 of treatment. The criteria for the two studies were the same through 12 weeks of treatment; the phase 2b study had more stringent criteria after 12 weeks.

The doses of LY2409021 used in the phase 2b study were selected on the basis of the exposure response analysis of a phase 1, 28-day, multiple-dose study that indicated that doses of LY2409021 ranging from 30 to 90 mg/day produced substantial lowering of fasting glucose levels in patients with type 2 diabetes (12) and the current phase 2a study that demonstrated reductions in fasting glucose and HbA1c levels in patients with type 2 diabetes at all dose levels (10, 30, and 60 mg). The doses of LY2409021 selected for the phase 2b study (2.5, 10, and 20 mg once daily) were used to identify the minimal efficacious dose and maximize the ability to demonstrate clinically meaningful efficacy with acceptable hepatic safety over the 24-week treatment period.

Study Participants

Patients were adults (aged 18–70 years, inclusive) with diagnoses of type 2 diabetes according to the World Health Organization diagnostic criteria (19) who had been treated with diet and exercise alone or in combination with a stable dose of metformin (≥1,000 mg/day) for at least 3 months before screening. Patients had HbA1c values of 6.5–10% (48–86 mmol/mol) and BMI between 25 and 40 kg/m2, inclusive (phase 2a study), and 7.0–10.5% (53–91 mmol/mol) and a BMI between 25 and 45 kg/m2, inclusive (phase 2b study). Patients agreed to self-monitor blood glucose (SMBG) levels, complete study diaries, and maintain consistent dietary, physical activity, and sleeping patterns throughout the study. Stable doses of antihypertensives or lipid-lowering medications were allowed. Clinical signs or symptoms of liver disease, previous diagnosis or serologic evidence of hepatitis B or C infection, and use of any antihyperglycemic medication other than metformin within the 3-month period before screening were exclusionary for both studies. Additional exclusion criteria at screening included aminotransferase levels greater than two times the ULN and fasting triglycerides >400 mg/dL in the phase 2a study and aminotransferase levels >2.5 times the ULN, nonfasting triglyceride levels >600 mg/dL, and use of insulin or glucagon-like peptide-1 (GLP-1) agonist for >5 days within the 3-month period before screening in the phase 2b study.

Outcome Measures

The primary efficacy end point was mean change in HbA1c level from baseline to end of the 12-week LY2409021 treatment period (phase 2a study) and to the end of the 24-week treatment period (phase 2b study). Secondary end points (in both studies) included changes in fasting serum glucose, glucagon, and GLP-1 (total and active [7–36]) levels and fasting serum insulin levels. Seven-point SMBG profiles consisted of blood glucose values obtained before and 2 h after each meal and at bedtime (phase 2a study) or at 0300 h (phase 2b study) on two separate days during the 5-day period before the respective study visits. For the phase 2a study, fasting lipid profiles were collected at initiation of treatment (week 0) and at end point (week 12); fasting body weight and sitting blood pressure measurements were collected before randomization and weekly throughout the treatment period. For the phase 2b study, fasting lipid profiles were collected at baseline and weeks 8, 12, 16, and 24; fasting body weight and sitting blood pressure measurements were collected before randomization and biweekly throughout the treatment period. Hepatic safety assessment included weekly (phase 2a study) or biweekly (phase 2b study) monitoring of ALT; aspartate aminotransferase (AST); γ-glutamyl transferase (GGT); alkaline phosphatase (ALK); and total, direct, and indirect bilirubin levels.

Analytical Methods

Blood samples for glucagon and GLP-1 analysis were collected in prechilled tubes containing EDTA. After collection, aprotinin/Trasylol (Bayer) (glucagon samples) and dipeptidyl peptidase-4 inhibitor (GLP-1 samples) additives were added as a preservative. For the phase 2a study, plasma glucagon concentrations were measured by radioimmunoassay by ALPCO Diagnostics (Salem, NH). For the phase 2b study, plasma glucagon concentrations were measured using an electrochemiluminescence sandwich immunoassay (20). Both assays have <1% cross-reactivity to oxyntomodulin or gut glucagon-like immunoreactivity. LY2409021 does not cross-react with immunoassays where glucagon and GLP-1 are measured. LY2409021 binds only to the receptor, not to ligands of the receptor or related receptors. For both phase 2a and 2b studies, total and active (7–36) plasma GLP-1 levels were measured using an ELISA (Millipore Corp., St. Charles, MO) and electrochemiluminescence sandwich immunoassay (Meso Scale Discovery, Rockville, MD), respectively; fasting serum glucose levels were measured using a commercially validated method; serum insulin levels were measured using direct chemiluminescent immunoassays (ADVIA Centaur; Siemens, New York, NY); patient 7-point SMBG levels were measured using a calibrated glucose meter; total cholesterol, HDL cholesterol, and triglyceride levels were determined directly and LDL cholesterol was calculated and systolic and diastolic blood pressure (SBP and DBP) were measured with an automated cuff while the patient was in a sitting position.

Data Analyses

For both phase 2a and 2b studies, efficacy analyses were conducted on the modified intent-to-treat population, defined as all randomized patients with at least one postbaseline measurement according to the treatment the patients were assigned. Mixed-effects model repeated measures (MMRM) analysis was used for the primary analysis of HbA1c levels for both studies. The least squares (LS) mean differences, 90% CIs, and P values reported were based on a model that included baseline HbA1c as a covariate and metformin use, visit, treatment, and visit-by-treatment interaction as fixed effects. Change in HbA1c level from baseline to end point was also analyzed using ANCOVA with baseline as a covariate and treatment and metformin use as fixed effects. There were no adjustments of multiplicity for any analyses unless otherwise stated. Missing end points were imputed using last observation carried forward. For the phase 2b study, categorical analyses on HbA1c were also performed to compare the proportions of patients who achieved HbA1c values ≤6.5% (48 mmol/mol) and <7.0% (53 mmol/mol) at week 24 by treatment group. A Cochran-Mantel-Haenszel test was used and stratified by baseline HbA1c values (<8.2%, ≥8.2% [66 mmol/mol]). For both phase 2a and 2b studies, analyses of secondary efficacy measures with continuous data were performed using similar statistical models. Fisher exact test also compared each treatment group with the placebo group for categorical outcomes. An ANCOVA was performed on the change from baseline aminotransferase values at each visit up to the end of the posttreatment washout period and analyzed using an MMRM model. The model consisted of the baseline value as a covariate and visit, treatment, and visit-by-treatment interaction as fixed effects. Treatment comparisons were reported as the treatment LS mean, 95% CI, and P value. Safety analyses were performed on the safety population, which was defined as all randomized patients who received at least one dose of the assigned study drug.

Patient Disposition, Demographics, and Baseline Characteristics

In the phase 2a study, 87 patients were randomized, 68 (78.2%) completed the study, and 21.8% of patients prematurely discontinued (Supplementary Fig. 2A). The most commonly reported reason for study discontinuation was subject decision (8.0%). Discontinuations due to adverse events (five patients, 5.9%) were driven by abnormalities in laboratory values (two events of liver aminotransferase level increases and three events of serum amylase and/or lipase level increases) without any clinical symptoms. All discontinuations due to adverse events occurred in patients receiving LY2409021. In the phase 2b study, 254 patients were randomized and 151 (59%) completed the study: 29 in the placebo group, 36 in the 2.5-mg group, 43 in the 10-mg group, and 43 in the 20-mg group (Supplementary Fig. 2B). Approximately 40% of patients (103 of 254) discontinued early. The most common reasons for early discontinuation were loss of glycemic control based on protocol discontinuation criteria (49 [19%]), protocol violation (24 [9%]), and subject decision (18 [7%]). Most of the per-protocol discontinuations due to inadequate glycemic control occurred at week 14 when hyperglycemia discontinuation criteria became more stringent. Specifically, eleven placebo patients (17%), eight 2.5-mg patients (13%), seven 10-mg patients (11%), and four 20-mg patients (6%) discontinued the study at week 14 because of protocol discontinuation criteria related to inadequate glycemic control. The mean and median days of treatment were 130 and 167, respectively.

Baseline characteristics for phase 2a study participants (Supplementary Table 1) showed that HbA1c values (mean, % [mmol/mol]) at week 0 for the LY2409021 treatment groups were highest in the 10-mg group (8.0% [64 mmol/mol]) and were 7.5% (58 mmol/mol) for the 30-mg group, 7.6% (60 mmol/mol) for the 60-mg group, and 7.9% (62 mmol/mol) for the placebo group. The most common concomitant medication was metformin, used by 58.6% of patients.

Baseline characteristics for phase 2b study participants (Supplementary Table 2) showed overall similarity in treatment group demographics. The mean HbA1c level was 8.0% (64 mmol/mol) at study entry. Concomitant metformin use was similar among the treatment groups (ranging from 85.7 to 87.5%).

Glucose-Related Efficacy Measurements

In the phase 2a study, after 12 weeks of treatment, LS mean (90% CI) change from baseline for HbA1c level in the three LY2409021 dose groups was significantly greater than that in the placebo group: –0.83% (–1.28, –0.38) (–9.1 mmol/mol) for the 10-mg group, –0.65% (–0.93, –0.37) (–7.1 mmol/mol) for the 30-mg group, and –0.66% (–1.00, –0.31) (–7.2 mmol/mol) for the 60-mg group (P = 0.03, P = 0.04, and P = 0.05, respectively). The change from baseline for the placebo group was 0.11% (–0.44, 0.65) (1.2 mmol/mol). The LS mean changes from baseline in HbA1c values for all LY2409021 dose groups were significantly greater compared with those of the placebo group from week 6 through week 12 (Fig. 1A). In the phase 2b study, after 24 weeks of treatment, 10 and 20 mg LY2409021 produced statistically significantly lower (P < 0.001) HbA1c values than placebo (Fig. 1B). The LS mean (90% CI) change from baseline for HbA1c value was –0.92% (–1.12, –0.73) (–10.1 mmol/mol) at 20 mg, –0.78% (–0.97, –0.58) (–8.5 mmol/mol) at 10 mg, –0.45% (–0.65, –0.25) (–4.9 mmol/mol) at 2.5 mg, and –0.15% (–0.37, 0.06) (–1.6 mmol/mol) for the placebo group. A small increase in HbA1c level was seen from week 12 to the end of the active treatment period (week 24) in the 10- and 20-mg groups (Fig. 1B). MMRM was used for the primary analysis of HbA1c levels for both studies. The ANCOVA (last observation carried forward) analysis demonstrated a greater magnitude of reduction in HbA1c level across treatment groups (not shown) than the MMRM analysis. Nonetheless, the results for the MMRM and ANCOVA analyses were generally similar.

Figure 1

Time course for LS mean (90% CI) change from baseline in HbA1c level by week and treatment with LY2409021 or placebo over the 12-week phase 2a study (A) and 24-week phase 2b study (B) treatment periods. Time course for LS mean (95% CI) change from baseline in fasting glucose level by week and treatment with LY2409021 or placebo over the 12-week phase 2a study (C) and 24-week phase 2b study (D) treatment periods. *P < 0.001 and †P < 0.05, compared with placebo. **P = 0.05, compared with placebo.

Figure 1

Time course for LS mean (90% CI) change from baseline in HbA1c level by week and treatment with LY2409021 or placebo over the 12-week phase 2a study (A) and 24-week phase 2b study (B) treatment periods. Time course for LS mean (95% CI) change from baseline in fasting glucose level by week and treatment with LY2409021 or placebo over the 12-week phase 2a study (C) and 24-week phase 2b study (D) treatment periods. *P < 0.001 and †P < 0.05, compared with placebo. **P = 0.05, compared with placebo.

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In the phase 2b study, one-third of the 20-mg treatment group achieved HbA1c levels ≤6.5% (48 mmol/mol; P < 0.001), and approximately half achieved HbA1c levels <7% (53 mmol/mol; P = 0.004) compared with the placebo (Supplemental Fig. 3B).

In the phase 2a study, LY2409021 produced statistically and clinically significant reductions in fasting glucose levels compared with placebo (Fig. 1C). The 30- and 60-mg dose groups showed reductions (P < 0.05) in fasting glucose levels that were sustained throughout the active treatment period. The LS mean change from baseline in fasting glucose levels for the phase 2b study is shown in Fig. 1D. LY2409021 produced dose-dependent, statistically significantly greater changes from baseline in fasting glucose levels than placebo; maximal decreases occurred within 2 weeks of dosing. A small increase in fasting glucose level was observed from week 12 to the end of the active treatment period (week 24) in the 10- and 20-mg dose groups (Fig. 1D).

In the phase 2a study, the means of 7-point SMBG levels (Fig. 2A) decreased from baseline in all three LY2409021 treatment groups at all pre- and postmeal times and at all visits but generally increased from baseline in the placebo group. In the phase 2b study, at baseline (week 0), all treatment groups had similar fasting, preprandial, postprandial, and 0300 h mean blood glucose values (Fig. 2B). By study end point (week 24), blood glucose levels were lower for the 10- and 20-mg LY2409021 doses than for the placebo group across the time points (Fig. 2B); the 20-mg group showed significantly lower glucose levels than the placebo group at most preprandial and postprandial time points.

Figure 2

LS mean (95% CI) change from baseline in SMBG level by time point and treatment with LY2409021 or placebo at end point week 12 (phase 2a study) (A) and week 24 (phase 2b study) (B). The SMBG level at each visit consisted of blood glucose values collected before and 2 h after each of the three main meals and at bedtime (phase 2a study) or at 0300 h (phase 2b study) as described in 2research design and methods. *P < 0.05, compared with placebo at indicated time points (A).*P < 0.001 and †P < 0.05, compared with placebo at indicated time points (B).

Figure 2

LS mean (95% CI) change from baseline in SMBG level by time point and treatment with LY2409021 or placebo at end point week 12 (phase 2a study) (A) and week 24 (phase 2b study) (B). The SMBG level at each visit consisted of blood glucose values collected before and 2 h after each of the three main meals and at bedtime (phase 2a study) or at 0300 h (phase 2b study) as described in 2research design and methods. *P < 0.05, compared with placebo at indicated time points (A).*P < 0.001 and †P < 0.05, compared with placebo at indicated time points (B).

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Non–Glucose-Related Pharmacodynamic Measurements

In both studies, dose-dependent increases in fasting glucagon levels were observed with LY2409021 treatment and appeared to plateau after ∼4 weeks of treatment (Fig. 3). Mean changes from baseline in fasting glucagon levels (Fig. 3A, phase 2a study) were largest in the 60-mg treatment group (4.5-fold increase at week 4). An apparent increase in fasting glucagon levels in the placebo group at weeks 6–10 was in part the result of a postanalytical error in a single patient’s samples. In the phase 2b study (Fig. 3B), the LS mean changes from baseline in fasting glucagon levels were largest in the 20-mg group, with a 4.5-fold increase at week 4 similar to that in the phase 2a study. Fasting glucagon levels returned to baseline in all treatment groups by the end of the posttreatment washout period (Fig. 3).

Figure 3

A: Time course for mean (±SE) change from baseline in fasting glucagon level (pmol/L) by week and treatment with LY2409021 or placebo over the 12-week phase 2a study treatment period. B: Time course for LS mean (95% CI) change from baseline in fasting glucagon level (pmol/L) by week and treatment with LY2409021 or placebo over the 24-week phase 2b study treatment period. C: Time course for mean (±SE) change from baseline in fasting total GLP-1 level (pmol/L) by week and treatment with LY2409021 or placebo over the 12-week phase 2a study treatment period. D: Time course for LS mean (95% CI) change from baseline in fasting total GLP-1 level (pmol/L) by week and treatment with LY2409021 or placebo over the 24-week phase 2b study treatment period. *P < 0.05, compared with placebo at indicated time points (A). *P < 0.001 and †P < 0.05, compared with placebo at indicated time points (B and D). No statistical analysis was conducted for fasting total GLP-1 level in C.

Figure 3

A: Time course for mean (±SE) change from baseline in fasting glucagon level (pmol/L) by week and treatment with LY2409021 or placebo over the 12-week phase 2a study treatment period. B: Time course for LS mean (95% CI) change from baseline in fasting glucagon level (pmol/L) by week and treatment with LY2409021 or placebo over the 24-week phase 2b study treatment period. C: Time course for mean (±SE) change from baseline in fasting total GLP-1 level (pmol/L) by week and treatment with LY2409021 or placebo over the 12-week phase 2a study treatment period. D: Time course for LS mean (95% CI) change from baseline in fasting total GLP-1 level (pmol/L) by week and treatment with LY2409021 or placebo over the 24-week phase 2b study treatment period. *P < 0.05, compared with placebo at indicated time points (A). *P < 0.001 and †P < 0.05, compared with placebo at indicated time points (B and D). No statistical analysis was conducted for fasting total GLP-1 level in C.

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LY2409021 treatment also led to dose-related higher levels of fasting total GLP-1 than placebo did; fasting total GLP-1 levels followed a pattern similar to that of fasting glucagon levels in both studies (Fig. 3C and D). Total fasting GLP-1 levels returned to baseline (Fig. 3C) during the posttreatment washout period in the phase 2a study (GLP-1 levels were not measured during washout in the phase 2b study). There were no statistically significant differences in fasting active GLP-1 levels between LY2409021 treatment at any dose and placebo for either study. Fasting insulin values were not significantly different between LY2409021 treatment and placebo (data not shown).

Safety and Tolerability

Dose-related, reversible increases in serum ALT levels were observed during active treatment with LY2409021 (Fig. 4). In the phase 2a study, 4 of 85 patients had ALT levels three or more times the ULN: 1 patient in the 30-mg group; 2 patients in the 60-mg group; and 1 patient in the 10-mg group, whose ALT level reached five or more times the ULN. There was no dose-dependent change from baseline in GGT (data not shown). There were no clinically significant changes in body weight, lipid levels, blood pressure, heart rate, or electrocardiogram measurements observed in the phase 2a study.

Figure 4

A: Time course for mean (±SE) change from baseline in ALT level (units/L) by week and treatment with LY2409021 or placebo over the 12-week phase 2a study treatment period. The ULNs for ALT level were 43 units/L (dashed line, male) and 34 units/L (dotted line, female). B: Time course for LS mean change (95% CI) from baseline in ALT level (units/L) by week and treatment with LY2409021 or placebo over the 24-week phase 2b study treatment period. Baseline ALT level = 32 units/L. *P < 0.05 and **P < 0.10, compared with placebo at indicated time points (A). *P < 0.05, compared with placebo at indicated time points (B).

Figure 4

A: Time course for mean (±SE) change from baseline in ALT level (units/L) by week and treatment with LY2409021 or placebo over the 12-week phase 2a study treatment period. The ULNs for ALT level were 43 units/L (dashed line, male) and 34 units/L (dotted line, female). B: Time course for LS mean change (95% CI) from baseline in ALT level (units/L) by week and treatment with LY2409021 or placebo over the 24-week phase 2b study treatment period. Baseline ALT level = 32 units/L. *P < 0.05 and **P < 0.10, compared with placebo at indicated time points (A). *P < 0.05, compared with placebo at indicated time points (B).

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In the phase 2b study, no placebo subjects and 8 of 191 patients receiving LY2409021 had ALT levels three or more times the ULN: 2 patients in the 2.5-mg group, 4 patients in the 10-mg group, and 2 patients in the 20-mg group. One patient in the 20-mg group had an ALT level five or more times the ULN. No subject with elevated ALT exhibited concomitant elevation of bilirubin in either study, and no clinical signs or symptoms of liver injury were reported. In all patients, the ALT levels returned to baseline either with continued treatment or after cessation of treatment (Fig. 4). Changes in AST levels showed a similar dose-related trend as changes in ALT levels but were less pronounced (data not shown). In the phase 2b study, GGT values increased beginning at week 2 and continued to rise through week 24 in the 20-mg group. During the 4-week posttreatment washout period, the GGT values of the 20-mg group began to reverse but were still above baseline values and statistically significantly greater than those for the placebo group (data not shown). There were no dose-dependent changes from baseline in ALK or in total, direct, or indirect bilirubin in either study (data not shown).

In the phase 2b study, there were no statistically significant differences in plasma lipid levels between any of the LY2409021 treatment groups and the placebo group. In 8 of 12 biweekly visits in the phase 2b study, there were no statistically significant differences in LS mean change from baseline in SBP or DBP between any of the LY2409021 treatment groups compared with the placebo group. On four visits, the LS mean change from baseline for SBP and DBP was significantly greater for one or more LY2409021 dose groups compared with the placebo group. After 24 weeks of treatment, only the 2.5-mg group showed a statistically significant difference in LS mean change from baseline to end point in SBP relative to the placebo group (LS mean difference, 4.9 mmHg; P = 0.038). The LS mean change from baseline in DBP for all three LY2409021 cohorts was <1 mmHg at week 24. Changes in body weight after 24 weeks of treatment were as follows: placebo, –1.07 kg; 2.5 mg, –0.33 kg; 10 mg, 0.55 kg; and 20 mg, 0.07 kg. Only the change for the 10-mg dose was statistically significantly different from the change for placebo (P = 0.033).

Adverse Events

There were no severe treatment-emergent adverse events (TEAEs) or deaths reported in either study. The percentages of patients who experienced any TEAE in the phase 2a study were similar across treatment groups (Supplementary Table 3). Nausea and hypoglycemia (4.7% of patients for each) were the most frequent TEAEs, but there was no dose dependency and no severe hypoglycemic events observed in any group. Two serious adverse events (SAEs) were reported: one subject in the 60-mg group had cellulitis, which was considered not related to study drug, and one patient in the 10-mg group had elevated ALT, AST, and GGT levels. The investigator considered the elevations related to study drug; however, the subject had undetectable levels of LY2409021 at the time of the event. Given the very long half-life of the study drug (∼60 h), it appears that the subject had not been recently compliant with treatment. Both SAEs were of moderate intensity and were resolved.

In the phase 2b study, the percentages of patients who experienced any TEAE (Supplementary Table 4) were slightly higher in the LY2409021 groups than in the placebo group. The most common TEAEs of all causes were ALT and AST level increases (6.7 and 5.1%, respectively) and headache (5.5%). A total of seven patients reported SAEs: two patients in the 10-mg group (AST level increased and atrial fibrillation), two patients in the 20-mg group (ALT level increased and AST level increased), and three placebo-treated patients (ankle fracture [one patient], cholecystectomy [one patient], and gastritis and iron deficiency anemia [one patient]). The three SAEs related to increased ALT and/or AST levels were all asymptomatic. The incidence of hypoglycemia (21) was low and not statistically different across drug treatment and placebo groups; 17 patients (7%) reported a total of 20 events (placebo, 2 patients; 2.5 mg, 4 patients; 10 mg, 6 patients; 20 mg, 5 patients). None of the cases was severe, and all cases were self-treated with an oral glucose source.

The role of insulin-glucagon imbalance continues to be of interest in understanding the pathophysiology of type 2 diabetes. Clinical data to support the potential utility of glucagon receptor antagonism in the treatment of diabetes, however, have lagged (10,22). Over 10 years has elapsed between publication of the first clinical study of a glucagon receptor antagonist (Bay 27-9955) (22) and the more recent studies of glucagon receptor antagonists LY2409021 (11,12), LY2786890 (13), MK-0893 (14,15), PF-06691874 (16), LGD-6972 (17), and ISIS-GCGRRX (18).

The current report details findings from two studies that together compose the largest and longest experience evaluating the safety and efficacy of a glucagon receptor antagonist published to date. We have shown that once-daily treatment with LY2409021 at doses of 10–60 mg for up to 24 weeks produced statistically significant and clinically relevant reductions in HbA1c levels and secondary efficacy measures in patients with type 2 diabetes without significantly increasing the risk of hypoglycemia. The improvements in glycemic parameters in the present studies are generally consistent with the findings of our previous studies with LY2409021 (12), including reduction in glycemia at all time points during SMBG testing. This suggests that glucagon signaling contributes to hyperglycemia in type 2 diabetes in both the fasting and postprandial states, as has been reported by others (9,23). The reductions in fasting glucose and HbA1c in the current studies are less pronounced than those that have been reported with some other glucagon receptor antagonists (14,18). This may be due, in part, to the fact that baseline HbA1c values were higher in those studies than in the current report. Glucose-lowering efficacy in the current studies is also likely to have been reduced, in part, by our efforts to find doses that are not associated with aminotransferase elevations. Pharmacokinetic-pharmacodynamic modeling using data from a study of LY2409021-mediated antagonism of exogenous hyperglucagonemia (11) suggests that the 20-mg dose used in the current phase 2b study would produce ∼67% blockade of the glucagon receptor at steady state, and maximal reductions in fasting glucose levels were consistently produced only at doses of 60 mg and higher in a previous study (12). Finally, the efficacy observed at the end of 24 weeks of treatment in the phase 2b study was attenuated by the slight rise in HbA1c and fasting glucose levels after week 12 in the 10- and 20-mg groups. Whether this reflects a waning of efficacy, an artifact of the higher discontinuation rate during this phase of the study, or something else is unclear.

LY2409021 produced dose-related increases in total GLP-1 levels that returned to baseline over the course of the 4-week posttreatment washout period. Consistent with our previous findings (12), levels of fasting active GLP-1 did not change significantly with LY2409021 treatment. Both glucagon and GLP-1 are derived from the proglucagon precursor, and the parallel increases in total GLP-1 and glucagon suggest the possibility that both glucagon and GLP-1 are secreted from pancreatic α-cells as a result of an endocrine feedback loop upon glucagon receptor blockade (24). Support for such a systemic feedback loop comes from results showing that liver-selective “knockout” of the glucagon receptor in mice can promote hyperglucagonemia (25). However, the acute nature of the increase in circulating glucagon also suggests that partial blockade of the glucagon receptor is most likely driven by a direct systemic feedback loop but might also be impacted by decreased overall glucagon clearance. Studies in mice have provided conflicting results regarding the role of GLP-1 in the improved glucose metabolism observed in glucagon receptor knockouts (26,27). Our results suggest that GLP-1 is unlikely to contribute to the efficacy of LY2409021 because active hormone levels are unchanged.

α-Cell hyperplasia has been raised as a potential concern with pharmacologic antagonism of glucagon action (28). Mice with homozygous disruption of the glucagon receptor gene develop dramatic elevations in plasma glucagon levels (>1,000-fold), hyperplasia of α-cells (29), and even neuroendocrine tumors (30). However, mice with heterozygous knockout of the glucagon receptor gene have very modest increases in glucagon and do not develop α-cell hyperplasia (29). In the present phase 2a and 2b studies, fasting glucagon was increased in a dose-dependent manner; maximum mean increases were ∼4.5-fold. The changes in glucagon levels seen with LY2409021 probably reflect a direct pharmacologic effect of glucagon receptor antagonism rather than an effect on α-cell number: the effect occurred early, was nonprogressive during treatment, and reversed completely during posttreatment washout.

Body weight, blood pressure, and lipid increases have been reported for other glucagon receptor antagonists (14,15). In the current studies, there were no significant changes in plasma lipid levels with LY2409021 treatment. Changes in body weight and blood pressure were generally small and not dose dependent, but some statistically significant differences from baseline were observed in the larger phase 2b study. These parameters will continue to be of key interest in future studies. A study to investigate the effect of LY2409021 on 24-h blood pressure profiles as measured by ambulatory blood pressure measurement is under way (31).

It is our view that hepatic safety remains the most important safety question facing the development of glucagon receptor antagonists for the treatment of type 2 diabetes. A key objective for both studies reported here was to evaluate the therapeutic margin between doses of LY2409021 that lower glucose levels and those that increase serum aminotransferase levels. We were unable to identify an efficacious dose of LY2409021 that was not associated with an increase in mean serum aminotransferase levels. However, changes in ALT and AST levels were nonprogressive over time, reversible with treatment discontinuation, modest in magnitude, and not associated with other signs or symptoms of hepatic dysfunction. Changes in serum aminotransferases observed with LY2409021 are probably related directly to antagonism of glucagon action; similar, reversible increases in aminotransferase levels have been seen in clinical studies with other small-molecule glucagon receptor antagonists (1417), with the human glucagon receptor monoclonal antibody LY2786890 (13), and with an antisense oligonucleotide targeting glucagon receptor gene expression (18). The cellular mechanisms by which these changes occur remain unknown. Complete abrogation of glucagon signaling has been reported to increase susceptibility of mice to experimental liver injury (32,33). Hepatic steatosis has also been suggested as a possible consequence of blocking glucagon action (33). However, results with pharmacologic or genetic disruption of glucagon signaling in rodents have been contradictory on this point (32,33,3537). In light of these unresolved questions, we have initiated a 12-month placebo- and active comparator–controlled hepatic safety study of LY2409021 that includes magnetic resonance imaging to assess potential changes in hepatic fat fraction (34). The results of this study could have significant implications for the future development of LY2409021 and possibly other glucagon receptor antagonists.

Clinical trial reg. nos. NCT01241448 (phase 2a) and NCT00871572 (phase 2b), clinicaltrials.gov.

Y.D. is currently affiliated with the Department of Biostatistics, University of Pittsburgh, Pittsburgh, PA, and C.N.L. is currently affiliated with the Department of Experimental and Clinical Pharmacology, University of Minnesota Twin Cities, Minneapolis, MN.

See accompanying article, p. 1075.

Acknowledgments. The authors thank the principal investigators and their clinical staff as well as the many study participants who generously agreed to participate in these clinical trials. The authors also thank the clinical operations staff for their excellent trial implementation and support. The authors thank Lakechie Turnipseed (Eli Lilly and Company) for contributions to study design, implementation, and management as the clinical trial manager for the phase 2a study. The authors also thank Dr. Robert Panek (INC Research, Raleigh, NC) who provided medical writing assistance.

Duality of Interest. This study was sponsored by Eli Lilly and Company and/or one of its subsidiaries. C.M.K., R.P.K., P.G., H.F., D.E.W., W.H.L., M.A.D., D.E.M., and T.A.H. are employees and stockholders of Eli Lilly and Company and/or one of its subsidiaries. Y.D. and C.N.L. were employees of Eli Lilly and Company and/or one of its subsidiaries. A.J.L. was an employee of the National Research Institute, Los Angeles, CA, at the time of this work and is currently retired. No other potential conflicts of interest relevant to this article were reported.

Author Contributions. For the phase 2a study, C.M.K., R.P.K., P.G., D.E.W., W.H.L., and M.A.D. contributed to the study concept and design, analysis and interpretation of data, and drafting of the manuscript. C.S., C.N.L., and H.F. contributed to drafting the manuscript and critical revisions. For the phase 2b study, C.M.K., R.P.K., P.G., D.E.W., A.J.L., D.E.M., and T.A.H. contributed to the study concept and design, analysis and interpretation of data, drafting the manuscript, and critical revisions. Y.D. contributed to the study analysis, interpretation of data, drafting the manuscript, and critical revisions. The authors certify that this manuscript represents valid work and that this manuscript has not been published and is not being considered for publication elsewhere. All authors contributed to the writing and review process and approved the final manuscript. C.M.K. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. The phase 2a study was presented in part in poster form at the 72nd Scientific Sessions of the American Diabetes Association, Philadelphia, PA, 8–12 June 2012, and at the 48th Annual Meeting of the European Association for the Study of Diabetes, Berlin, Germany, 1–5 October 2012. The phase 2b study was presented in part in poster form at the 73rd Scientific Sessions of the American Diabetes Association, Chicago, IL, 21–25 June 2013.

Acknowledgments. The authors thank the principal investigators and their clinical staff as well as the many study participants who generously agreed to participate in these clinical trials. The authors also thank the clinical operations staff for their excellent trial implementation and support. The authors thank Lakechie Turnipseed (Eli Lilly and Company) for contributions to study design, implementation, and management as the clinical trial manager for the phase 2a study. The authors also thank Dr. Robert Panek (INC Research, Raleigh, NC) who provided medical writing assistance.

Duality of Interest. This study was sponsored by Eli Lilly and Company and/or one of its subsidiaries. C.M.K., R.P.K., P.G., H.F., D.E.W., W.H.L., M.A.D., D.E.M., and T.A.H. are employees and stockholders of Eli Lilly and Company and/or one of its subsidiaries. Y.D. and C.N.L. were employees of Eli Lilly and Company and/or one of its subsidiaries. A.J.L. was an employee of the National Research Institute, Los Angeles, CA, at the time of this work and is currently retired. No other potential conflicts of interest relevant to this article were reported.

Author Contributions. For the phase 2a study, C.M.K., R.P.K., P.G., D.E.W., W.H.L., and M.A.D. contributed to the study concept and design, analysis and interpretation of data, and drafting of the manuscript. C.S., C.N.L., and H.F. contributed to drafting the manuscript and critical revisions. For the phase 2b study, C.M.K., R.P.K., P.G., D.E.W., A.J.L., D.E.M., and T.A.H. contributed to the study concept and design, analysis and interpretation of data, drafting the manuscript, and critical revisions. Y.D. contributed to the study analysis, interpretation of data, drafting the manuscript, and critical revisions. The authors certify that this manuscript represents valid work and that this manuscript has not been published and is not being considered for publication elsewhere. All authors contributed to the writing and review process and approved the final manuscript. C.M.K. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. The phase 2a study was presented in part in poster form at the 72nd Scientific Sessions of the American Diabetes Association, Philadelphia, PA, 8–12 June 2012, and at the 48th Annual Meeting of the European Association for the Study of Diabetes, Berlin, Germany, 1–5 October 2012. The phase 2b study was presented in part in poster form at the 73rd Scientific Sessions of the American Diabetes Association, Chicago, IL, 21–25 June 2013.

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Supplementary data