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Diabetes, Obesity and Metabolism 13: 426 – 433, 2011. © 2011 Blackwell Publishing Ltdoriginal article

A 5-week study of the pharmacokinetics and pharmacodynamics of LY2189265, a novel, long-acting glucagon-like peptide-1 analogue, in patients with type 2 diabetes P. Barrington1, J. Y. Chien2, H. D. H. Showalter2, K. Schneck2, S. Cui3, F. Tibaldi4, B. Ellis2 & T. A. Hardy2 1Eli Lilly and Company, Erl Wood, Surrey, UK 2 Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN, USA 3 i3StatProbe, Austin, TX, USA 4 GSK Biologicals, Rixensart, Belgium

Aim: To investigate the safety, tolerability, pharmacokinetics and pharmacodynamics of LY2189265 (LY), a novel, long-acting glucagen-like peptide-1 analogue, administered once weekly to subjects with type 2 diabetes. Methods: This was a placebo-controlled, parallel-group, subject- and investigator-blind study of LY in subjects (N = 43) with type 2 diabetes mellitus controlled with diet and exercise alone or with a single oral antidiabetic medication. Subjects taking metformin or thiazolidinediones continued on their therapy. Subjects receiving sulfonylurea, acarbose, repaglinide or nateglinide were switched to metformin prior to enrollment. Subjects received five once-weekly doses of 0.05, 0.3, 1, 3, 5 or 8 mg. Effects on glucose, insulin and C-peptide concentrations were determined during fasting and following standard test meals. The pharmacokinetics of LY and its effects on HBA1c, glucagon, body weight, gastric emptying and safety parameters were assessed. Results: Once-weekly administration of LY significantly reduced (p < 0.01) fasting plasma glucose, 2-h post-test meal postprandial glucose and area under the curve (AUC) of glucose after test meals at doses ≥1 mg. These effects were seen after the first dose and were sustained through the weekly dosing cycle. Most doses produced statistically significant increases in insulin and C-peptide AUC when normalized for glucose AUC. Statistically significant reductions in HBA1c were observed for all dose groups except 0.3 mg. The most commonly reported adverse effects (AEs) were nausea (35 events), headache (20 events), vomiting (18 events) and diarrhoea (8 events). Conclusions: LY showed improvement in fasting and postprandial glycaemic parameters when administered once weekly in subjects with type 2 diabetes. The pharmacokinetics and safety profiles also support further investigation of this novel agent. Keywords: antidiabetic drug, clinical trial, diabetes mellitus, GLP-1 analogue, pharmacodynamics, type 2 diabetes

Date submitted 31 March 2010; date of first decision 9 May 2010; date of final acceptance 4 January 2011

Introduction Therapeutic use of native glucagen-like peptide-1 (GLP-1) in the treatment of patients with type 2 diabetes mellitus is impractical because of its rapid inactivation by the protease dipeptidyl peptidase-IV (DPP-IV), resulting in a plasma half- life of 1 – 2 min [1]. As a consequence, multiple approaches have been used to develop pharmaceuticals that preserve the pharmacology of native GLP-1 while providing an extended duration of action. These include efforts to raise endogenous GLP-1 levels through the use of DPP-IV inhibitors and the development of GLP-1 receptor agonists that are resistant to DPP-IV inactivation. Exendin-4 (exenatide) is a naturally occurring GLP-1 receptor agonist which was the first GLP-1

Correspondence to: Dr. Philip Barrington, Eli Lilly and Company, Erl Wood Manor, Windlesham, Surrey GU20 6PH, UK. E-mail: [email protected]

mimetic approved for use in type 2 diabetes patients. Exenatide has been shown to restore first-phase insulin response [2], improve glycaemic control and promote weight loss in diabetic patients [3]. Despite resistance to DPP-IV cleavage, exenatide exhibits a relatively short half-life (2.4 h) which may be due, in part, to renal clearance of this peptide [4]. The limited duration of action necessitates twice-daily subcutaneous injections and produces a glucose lowering benefit that is primarily postpran- dial [3]. More recently, longer-acting GLP-receptor agonists have been developed that can be given as once-daily or even once-weekly injections. In addition to greater convenience, such agents may provide greater efficacy as a result of sustained pharmacology. Results from recent clinical trials with long- acting formulations of exenatide [5] and novel GLP-1 receptor agonists [6,7] supported the hypothesis for this class of agents.

LY is a GLP-1 analogue covalently linked to a constant fragment (Fc) of a human immunoglobulin class 4 (IgG4) [8]. Modification of the GLP-1 analogue amino acid sequence

DIABETES, OBESITY AND METABOLISM original article renders the molecule less susceptible to DPP-IV hydrolysis, while addition of the Fc fragment reduces renal clearance because of the size of the resultant fusion protein (molecular weight = 59.7 kDa) being less susceptible to glomerular filtration [8]. Both effects contribute to the prolonged time action of LY. Additional modifications have been introduced to make LY more soluble and less immunogenic [8]. A prior study of single, subcutaneous doses administered to healthy subjects showed that LY was safe and resulted in enhanced glucose-dependent insulin secretion and improved glucose tolerance in healthy volunteers [9]. Furthermore, the pharmacokinetic half-life of LY following single doses suggested that this molecule could be administered once weekly [9].

The primary objective of this study (study H9X-MC-GBCD) was to assess the safety and tolerability of multiple doses of LY in subjects with type 2 diabetes. The additional objectives were to determine the pharmacokinetic and pharmacodynamic effects of LY and to assess dose – exposure – response relation- ships. Key pharmacodynamic endpoints included changes in fasting glucose and glucose excursions after standardized test meals. Effects on HBA1c, body weight, insulin, glucagon and gastric emptying were also assessed.

Materials and Methods Study Design

This was a phase 1b, multi-center, placebo-controlled, subject- and investigator-blind adaptive study performed in males and females between the ages of 30 and 65 with type 2 diabetes.

The study was approved by the appropriate ethical review boards (Aspire IRB, La Mesa, CA, USA; RCRC IRB, Austin, TX, USA and Independent Investigational Review Board, Inc., Plan- tation, FL, USA) and conducted in accordance with the Helsinki Declaration and Good Clinical Practice. Subjects had a history of type 2 diabetes adequately controlled with diet or exercise alone or on stable doses of one of the following classes of oral antihyperglycaemic medications (OAM): metformin, sulfony- lureas, glinides, thiazolidinediones or acarbose. Subjects were tested for the presence of glutamate decarboxylase and islet cell antibodies at screening, thereby excluding subjects with type 1 diabetes. Subjects being treated with metformin or thiazolidine- diones at study entry were continued on that medication. Those receiving sulfonylureas, acarbose, repaglinide or nateglinide were switched to metformin, 500 – 1000 mg twice daily, for ≥3 weeks prior to randomization. Other key inclusion criteria included HBA1c 6.5 – 10.5% (subjects receiving an OAM and having an HBA1c of 6.1 – 6.5% were eligible to participate in the study provided they stopped the OAM ≥2 weeks prior to study drug administration), fasting glucose 108 – 250 mg/dl and body mass index (BMI) 25 – 40 kg/m2. Subjects with uncontrolled hypertension, history of autonomic neuropathy or conditions that could alter gastric emptying were excluded. Concomi- tant use of systemic corticosteroids or medications reducing gastrointestinal (GI) motility was excluded.

After providing written informed consent, eligible subjects were assigned to one of six cohorts. In each cohort, four subjects were randomized to LY or placebo in a 3 : 1 ratio. LY

doses studied were 0.05, 0.3, 1, 3, 5 and 8 mg; each adminis- tered once weekly for 5 weeks. Study drug was administered subcutaneously into the abdominal wall on study days 1, 8, 15, 22 and 29. The fixed dosing regimen was chosen to fully evaluate the adverse effect (AE) profile without dose titration, as the long-acting pharmacokinetic profile of LY was expected to show reduced incidence of GI side effects.

Cohorts were studied sequentially in a dose escalating fashion. An adaptive allocation procedure based on a modified continual reassessment method [10] was implemented to guide the assessment of the maximum tolerated dose (MTD). The adaptive algorithm included an assessment of the tolerability of all prior doses. Dose escalation continued until the MTD was estimated between 5 and 8 mg based on the incidence rates of nausea and vomiting. After the range of MTD was estimated, additional cohorts of subjects were allocated to lower doses (0.3 and 1 mg) based on analysis of interim data to refine the dose response for fasting and postprandial plasma glucose.

Pharmacokinetic and Pharmacodynamic Methods

Blood samples for pharmacokinetic (PK) assessment of LY were collected at pre-dose, 12, 24, 48 and 72 h after the first dose; at pre-dose between the second and the fourth dose and at pre- dose, 24, 48, 72, 144, 168, 264 and 336 h after the last (fifth) dose in week 5. Plasma concentrations of LY were assessed using a validated radioimmunoassay with lower and upper limits of quantification of 5.0 and 40.0 ng/ml, respectively. LY PK parameters were estimated using non-compartmental methods implemented in Pharsight WinNonlin® (Sunnyvale, CA, USA).

Pharmacodynamic assessments included plasma glucose, insulin and C-peptide concentrations measured under fasting conditions and after standardized test meals. Test meals were performed on day −1, 3, 31 and 36 and involved a solid meal of approximately 400 kcal comprised of 55% carbohydrates, 30% fat and 15% protein; plasma glucose values were checked prior to the meal and 0.25, 0.5, 1, 2, 3 and 4 h after the start of the meal. Tests on days 3 and 31 were intended to coincide with maximum drug concentrations after the first and fifth doses, respectively. The test meal on day 36 was included to evaluate the PD effect of LY at minimum drug concentration at the end of a weekly dosing cycle. The test meals were performed after an overnight fast. Plasma glucagon was determined dur- ing fasting and 2 h after initiating the test meal on day 36. Gastric emptying was assessed by following the absorption of acetaminophen into plasma following a single dose (480 mg) of acetaminophen elixir given with test meals on day −1 and at maximum drug concentration on day 3.

Plasma glucose, insulin, C-peptide, glucagon and acetaminophen concentrations were determined at a cen- tral laboratory using validated assays. Acetaminophen PK parameters were calculated using non-compartmental methods implemented in WinNonlin®.

Safety assessments included physical examination, vital sign measurements, laboratory tests, 12-lead ECGs and monitoring of AEs. Serum was tested for antibodies to LY prior to the first dose of study drug and on study days 31, 57 and 89 using a bridging-type enzyme-linked immunosorbent assay.

Volume 13 No. 5 May 2011 doi:10.1111/j.1463-1326.2011.01364.x 427

original article DIABETES, OBESITY AND METABOLISM Statistical Methods

For the analyses of glucose, HBA1c, weight, insulin, C-peptide parameters and acetaminophen PK, change from pre-treatment baseline was compared between active dose groups and placebo using mixed-effects linear models with baseline as covariate, treatment, time and treatment-by-time as fixed effects. The area under the curve for glucose (gAUC), insulin, C-peptide and acetaminophen were calculated using linear trapezoidal methods. Effects on glucagon were assessed based on postmeal values. LY pharmacokinetic parameters were summarized by dose group. Time to PK steady state was determined by comparing plasma drug concentrations prior to each dose to the concentration on day 36 using a step-down trend test. The variability of the PK parameters were assessed using a mixed linear model with treatment and week as fixed effects and subject as random effect.

The dose – concentration – response relationship for fasting glucose prior to standard test meals was analysed using non- linear mixed effects models implemented in NONMEM® version 6 (NONMEM Project Group, UCSF, San Francisco, CA, USA). The maximum glucose lowering effect (Emax ) and the dose to achieve 50% of maximum effect (ED50) were estimated.

Blood pressure, pulse rate and ECG intervals were analysed by mixed linear models with treatment, time and treatment-by-time as fixed effects and subject as random effect. Blood pressure and pulse rate analy- ses included baseline as covariate. The analyses of QT interval included baseline and RR interval as covari- ates, which was similar to Dmitrienko and Smith’s method [11]. Bazzett’s and Fridericia’s corrections were also performed.

All tests of treatment effects were conducted at a two-sided alpha level of 0.05 unless otherwise stated. Subjects that received at least one dose of study drug were included in analyses. Sta- tistical analyses were conducted using SAS version 8.2 (SAS Institute Inc., Cary, NC, USA) for Windows.

Results Demographics and Disposition

The study population was composed of 43 subjects with type 2 diabetes: 28 females and 15 males, approximately 42% Hispanic, 35% Caucasion, 16% Asian and 7% African American. Subjects ranged in age from 38 to 65 years. The mean BMI at baseline was 31 kg/m2 (range: 25 – 39 kg/m2) and the mean HBA1c was 7.6% (range 6.1 – 10.2%). Further demographic information including baseline fasting plasma glucose (FPG) and antidiabetic medications at screening are included in Table 1.

Among the 43 subjects randomized to treatment, 32 received at least one dose of LY and 11 received placebo. On the basis of the adaptive dose allocation procedure, the number of LY- treated subjects completing the study were three subjects on 0.05 mg, six subjects on 0.3 mg, four subjects on 1 mg, two subjects on 3 mg, eight subjects on 5 mg and five subjects on 8 mg. One subject withdrew from each of the 1, 3, 5 and 8 mg LY dose groups.

Pharmacokinetics

Mean plasma concentrations of LY as a function of time and dose are shown in figure 1. Assessment of trough LY concen- trations indicated that steady-state concentration levels were obtained after the second weekly dose with an average accu- mulation ratio (AUCweek 5: AUCweek 1) estimated to be 1.44. The average peak-to-trough concentration ratio (Cmax : Cmin ) was between two and three. The time to maximal plasma drug concentration (tmax) ranged from 12 to 72 h and was similar between the first and the fifth dose. The mean plasma half- life determined after the last dose was 95.4 h (approximately 4 days). The mean steady-state apparent clearance and volume of distribution were 0.157 l/h and 21.5 l, respectively.

The intra-subject variability for Cmax and AUC(0 – 168) were 29.4 and 14.6%, respectively. The intersubject variability for Cmax and AUC(0 – 168) were 28.5 and 30.8%, respectively. The majority of the plasma concentration data at the 0.05 mg dose were below the quantifiable limit.

Pharmacodynamics

Mean changes from baseline in FPG and 2-h postprandial glu- cose (PPG) after standardized test meals are shown in figure 2. LY treatment was associated with robust responses in FPG (figure 2A) and 2-h PPG (figure 2B), as well as the area under the plasma glucose-time curve (gAUC) (Appendix Table A1). Reductions in FPG were statistically significant (p < 0.05) at 0.05, 1, 3, 5 and 8 mg doses of LY. A positive dose response of LY on FPG (prior to standard test meals) was showed (p < 0.01) (figure 2A). The estimated maximum reduction in FPG was approximately 31% of the baseline and the esti- mated ED50 was 0.45 mg (SE = 0.57). Reductions in 2-h PPG and gAUC were statistically different (p < 0.05) from placebo at doses of 1 mg of LY and higher. Figure 2C, D showed the mean change from baseline in FPG and 2 h PPG val- ues for the three postbaseline days on which test meals were performed. Glycaemic reductions were apparent at the first assessment on day 3 (approximately 48 h after the first dose) and these were either sustained or showed further reduction when retested approximately 48 h after the last dose on day 31. These reductions were generally maintained throughout the weekly dosing cycle as seen by comparing the results of days 36 to 31.

Changes in HBA1c and body weight were secondary pharmacodynamic endpoints. HBA1c data were collected from 39 subjects that completed 5 weeks of treatment. In agreement with the observed changes in FPG and PPG, dose-dependent decreases in HBA1c occurred with LY treatments (figure 3A). Compared to placebo, reductions in HBA1c were statistically significant at all doses of LY except the 0.3 mg dose. A reduction in HBA1c was observed in only 3 of the 11 subjects treated with placebo, while 26 of the 28 subjects receiving LY showed a decrease from baseline. Mean reductions from baseline of approximately 1% were observed after 5 weeks of treatment at higher doses. Change in body weight from baseline to week 5 of treatment is shown in figure 3B. Weight loss was seen with LY treatment, but only at the highest doses in this 5-week study. Weekly dosing with 5 and 8 mg for 5 weeks led to

428 Barrington et al. Volume 13 No. 5 May 2011

DIABETES, OBESITY AND METABOLISM original article Table 1. Baseline demographics.

Placebo N = 11

LY2189265 0.05 mg N = 3

LY2189265 0.3 mg N = 6

LY2189265 1 mg N = 5

LY2189265 3 mg N = 3

LY2189265 5 mg N = 9

LY2189265 8 mg N = 6

Overall N = 43

Age (years) Mean 56 53.3 52.5 53.4 60 55.1 57.5 55.3 SD 6.1 2.3 4.3 8.7 2.6 8.2 3.5 6.1

Sex Male 6 0 2 1 0 6 0 15 Female 5 3 4 4 3 3 6 28

Race/ethnicity Caucasian 4 0 4 1 0 3 3 15 African 0 0 1 1 0 1 0 3 Hispanic 5 2 1 3 3 2 2 18 East Asian 2 1 0 0 0 3 0 6 West Asian 0 0 0 0 0 0 1 1

Weight (kg)∗ Mean 83.2 81.4 85.7 89.5 67.0 88.6 76.8 83.3 SD 8.0 10.0 12.2 18.8 12.3 15.6 8.2 13.0

Body mass index (kg/m2) Mean 29.9 34.1 31.8 32.3 28.3 30.6 30.1 30.8 SD 4.1 2.2 3.2 5.5 3.7 3.8 4.2 4.0

Fasting plasma glucose† Mean 158.4 170.7 185.5 200.4 145.2 142.5 177.3 166.3 SD 33.8 5.6 48.0 70.1 17.5 35.0 42.5 43.1

HBA1c (%) Mean 7.4 8.1 8.2 8.4 7.0 7.4 7.3 7.6 SD 1.0 1.0 1.3 1.8 0.4 0.7 0.8 1.1

Antidiabetic medications No medication 1 1 3 2 1 2 3 13 Metformin 7 2 3 3 2 7 2 26 Thiazolidinediones 1 0 0 0 0 0 1 2 SU changed to metformin 2 0 0 0 0 0 0 2

HBA1c, hemoglobin A1c; N, number of subjects; SD, standard deviation; SU, sulfonylurea. ∗Day 1 weights. †Day −1 glucose measurements.

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mean reductions in body weight of 2.5 and 2 kg, respectively. These changes were statistically significant compared to placebo (p < 0.05).

Mechanistic assessments included determination of insulin secretory response and plasma glucagon response to test meals. Statistically significant increases in insulin secretory parameters were observed at some doses of LY. For example, change from baseline in insulin AUC and C-peptide AUC after week 5 test meals were significantly greater than placebo for LY doses of 0.3, 5 and 8 mg. Changes in these parameters were not clearly dose-dependent because of small sample size, but the changes did reflect the dose-responsive improvement in glycaemic parameters noted above. Treatment effects on insulin secretory parameters were more apparent when changes in insulin or C-peptide were normalized relative to changes in glucose (Appendix figure A1). For example, the ratio of insulin AUC to gAUC increased in a dose-dependent manner. These changes were statistically significant (p < 0.05) compared to placebo at all doses of LY except the 0.05 and 3 mg doses. Glucagon levels were tested under fasting conditions and 2 h after the standard test meal on day 36. There was no consistent pattern regarding glucagon excursions during the test meal and no apparent dose-related changes in these parameters, perhaps because of the small sample size of this study (Appendix Table A2).

Comparison of acetaminophen PK given with the solid test meal on day −1 and 3 was used to assess the effect of LY on gastric emptying rate. Reductions in acetaminophen exposure

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original article DIABETES, OBESITY AND METABOLISM

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Figure 2. The least squares mean (LSM) changes from baseline in fasting plasma glucose (A) and 2-h postprandial glucose (PPG) (B) after the test meal following the fifth weekly dose of LY are shown (measurements on day 31 and 36 were averaged for the calculation of week 5 values that were compared to baseline values). Baseline values (day −1 glucose measurements) for fasting plasma glucose are available for each dose in Table 1. Mean baseline values for 2-h PPG are 233 mg/dl for placebo, 262 mg/dl for 0.05 mg, 245 mg/dl for 0.3 mg, 265 for 1 mg, 200 mg/dl for 3 mg, 209 mg/dl for 5 mg and 242 mg/dl for 8 mg. The mean 2-h PPG for all subjects was 235 mg/dl. Fasting (C) and 2-h PPG (D) were measured following a standard test meal 3 days (day 31) and 8 days (day 36) after the fifth weekly dose of LY. There were no statistically significant differences between changes observed on days 31 and 36.

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[Cmax and AUC(0 – 12)] ranged from 10 to 60%. A statistically significant (p < 0.01) difference in the change from baseline in the least square mean ratio of pre- to post-dose acetaminophen PK parameters relative to placebo was detected at the 5 mg dose for both Cmax (relative change of 0.41; 90% CI: 0.31, 0.53) and AUC(0 – 12) (relative change of 0.52; 90% CI: 0.44,

0.62). The maximum average delay in acetaminophen tmax was approximately 1.5 h, with a statistically significant difference observed in the change in the least squares mean ratio of pre- to post-dose measurements of tmax relative to placebo for the 8 mg dose of LY (relative change of 2.31; 90% CI: 1.28, 4.18).

430 Barrington et al. Volume 13 No. 5 May 2011

DIABETES, OBESITY AND METABOLISM original article Table 2. LSM change from baseline in vital signs compared to placebo.

Change in supine pulse rate (bpm) (90% CI) p-value

Change in supine systolic BP (mmHg) (90% CI) p-value

Change in supine diastolic BP (mmHg) (90% CI) p-value

LY2189265 dose Week 1 Week 5 Week 1 Week 5 Week 1 Week 5

Placebo∗ 3.59∗ 1.35∗ −2.87∗ 1.86∗ 1.01∗ 2.41∗ 0.05 mg −1.20 −0.29 6.28 0.18 3.26 1.67

(−5.16, 2.77) (−5.26, 4.69) (−1.59, 14.14) (−9.16, 9.52) (−1.57, 8.08) (−4.13, 7.47) p = 0.62 p = 0.92 p = 0.19 p = 0.97 p = 0.26 0.63

0.3 mg 1.02 0.07 4.18 −2.16 2.61 0.77 (−2.31, 4.34) (−3.59, 3.73) (−2.75, 11.12) (−9.53, 5.20) (−1.65, 6.87) (−3.76, 5.30) p = 0.61 p = 0.97 p = 0.31 p = 0.62 p = 0.31 p = 0.78

1 mg 3.08 3.98 1.97 −0.57 1.05 0.00 (−0.32, 6.47) (0.32, 7.63) (−4.93, 8.87) (−7.75, 6.60) (−3.19, 5.29) (−4.44, 4.44) p = 0.14 p = 0.07 p = 0.63 p = 0.89 p = 0.68 p = 1.00

3 mg 0.68 6.85 1.54 −2.72 0.32 3.32 (−3.61, 4.96) (1.72, 11.97) (−7.46, 10.54) (−12.82, 7.38) (−5.12, 5.75) (−2.85, 9.49) p = 0.79 p = 0.03 p = 0.77 p = 0.65 p = 0.92 p = 0.37

5 mg 5.71 9.87 7.12 1.26 2.80 1.36 (2.78, 8.65) (6.58, 13.17) (1.02, 13.23) (−5.30, 7.82) (−0.95, 6.55) (−2.69, 5.41) p < 0.01 p < 0.01 p = 0.06 p = 0.75 p = 0.22 p = 0.58

8 mg 0.91 8.99 4.73 1.33 0.64 0.67 (−2.46, 4.29) (5.10, 12.89) (−2.20, 11.66) (−6.28, 8.94) (−3.68, 4.97) (−4.09, 5.43) p = 0.65 p < 0.01 p = 0.26 p = 0.77 p = 0.80 p = 0.81

BP, blood pressure; LSM, least squares mean. ∗Estimate of the LSM change from baseline for placebo.

Safety

Safety analyses included all 43 subjects that received at least 1 dose of LY or placebo. Thirty-nine subjects completed the study. Four subjects were withdrawn from the study; three because of AE(s). Two subjects (one at a 1 mg dose and the other an 8 mg dose) were withdrawn because of nausea and vomiting related to LY. A third subject (at a 5 mg dose) was withdrawn because of increased eosinophil count and creatinine levels (thought to be related to dehydration); the increase was deemed unrelated to LY as eosinophil count decreased after a second dose of LY, administered prior to availability of the lab results from the first dose and before the patient was withdrawn.

One serious AE was reported during the study: a case of chest pain beginning approximately 60 h after the first dose of 8 mg LY. This was determined to be non-cardiac, but rather of gastrointestinal aetiology. The subject continued on study drug after the event resolved.

For subjects receiving LY, nausea (35 AEs in 16 subjects), headache (20 AEs in 10 subjects), vomiting (18 AEs in 8 subjects), diarrhoea (8 AEs in 6 subjects) and dizziness (7 AEs in 7 subjects) were the most commonly reported AEs and most AEs were of mild or moderate severity. With the exception of one subject who seemed particularly sensitive to the GI side effects of 1 mg LY, all other patients who experienced at least one episode of emesis were in the 5 mg (44% of subjects) or 8 mg cohorts (50% of subjects). In subjects who experienced AEs of nausea and/or vomiting, the majority of the events occurred after the first dose of LY. There was one AE of hypoglycaemia in a subject receiving 0.3 mg of LY who experienced shakiness with capillary blood glucose of 68 mg/dl.

Increases in supine pulse rate were observed at doses of 1 mg or greater, with the greatest change at the 5 mg dose. The

peak effect on pulse rate was observed 36 to 72 h post-dose. Values for least squares mean changes from baseline compared to placebo for pulse rate, supine diastolic and systolic blood pressure are presented in Table 2. LY was not associated with prolongation of corrected QT interval.

No subject developed antibodies to LY during the study and there were no apparent treatment-related changes in safety laboratory parameters.

Conclusions The current study evaluated the safety, pharmacokinetics and pharmacodynamic effects of a novel long-acting GLP- 1 analogue in subjects with type 2 diabetes. LY was found to be safe and generally well tolerated up to the range of MTD (between 5 and 8 mg). The AEs reported in this study are consistent with those reported following administration of other GLP-1 analogues [12]. Gastrointestinal AEs were most often mild to moderate in severity, although two subjects discontinued the study early because of nausea and vomiting. We also observed a modest increase in pulse rate with LY treatment, consistent with changes which have been reported with other GLP-1 analogues [13]. The nature of this relationship is unclear. Additional data in larger numbers of subjects is needed to assess the effect of LY on pulse rate and will be obtained in ongoing phase 2 and 3 studies. On the basis of the AE profile, vital signs and pharmacodynamic response observed in this phase 1 study, doses of LY upto 3 mg, were selected for further dose-response evaluation in phase 2 [14].

LY treatment was associated with statistically significant improvements in both FPG and PPG in this study. These effects were apparent as early as 24 h after the first dose. The glucose- lowering effects were also well maintained throughout the

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original article DIABETES, OBESITY AND METABOLISM 7-day dosing cycle. LY has a plasma half-life of approximately 4 days and steady-state trough concentrations of drug in the plasma were reached as early as the second week of treatment. A mean reduction in HBA1c of upto 1% was observed in this study despite only 5 weeks of treatment.

A key advantage that is more specific to GLP-1 analogues relates to the weight loss seen with this class of agents. Weight loss was observed in this study to a degree similar to what has been reported with other long-acting GLP-1 analogues in short-term studies [6,7]; however, it was only observed at the 5 and 8 mg dose levels. Similar weight loss may be expected at lower doses with longer treatment [14].

Preclinical studies with LY focused on the insulinotropic effects of this GLP-1 analogue [8]. A study in healthy vol- unteers has provided further evidence for glucose-dependent stimulation of insulin secretion by this agent [9]. This study showed statistically significant increases in plasma insulin or C-peptide at some doses of LY and this treatment effect was more apparent if these parameters were expressed relative to changes in plasma glucose. However, this study did not show a clear effect of LY on glucagon secretion or gastric emptying. This is somewhat surprising given these effects are consid- ered important contributors to the glucose-lowering effects of GLP-1 [3,15 – 19]. The relative contribution of these mecha- nisms may differ with continuous GLP-1 receptor agonism, as is expected with long-acting GLP-1 analogues. For example, a once-weekly formulation of exenatide appears to have less of an effect on gastric emptying than does exenatide given twice daily [5]. However, the lack of clear effects of LY on glucagon secretion or gastric emptying in this study may sim- ply reflect the small numbers of subjects tested and limitations of the measurements. Greater understanding of mechanistic determinants of LY action requires further study.

In conclusion, LY is a novel GLP-1 analogue covalently linked to a human IgG4 which retains key aspects of GLP-1 phar- macology and displays a pharmacokinetic profile in humans that supports a once-weekly dosing regimen. Weekly doses of LY were generally well tolerated and provided significant gly- caemic benefit to subjects with type 2 diabetes. Modest weight loss with higher doses was also seen in this short-term study. The safety, tolerability and sustained glucodynamic control afforded by weekly dosing of LY warrants further development of this agent as a once-weekly treatment for type 2 diabetes.

Acknowledgements This study was supported by Eli Lilly and Company. All authors, except F. Tibaldi and S. Cui, are current employees of Eli Lilly and Company. F. Tibaldi is a former employee of Eli Lilly and Company and current employee of GSK. S. Cui is an employee of i3StatProbe. All authors are stock owners in Eli Lilly and Company with the exception of Dr F. Tibaldi and Dr S. Cui.

The authors would like to acknowledge Brenda Gaydos for her contribution to the study design. The authors would like to thank the investigators in this trial: Dr Mark Kipnes (Diabetes & Glandular Disease Research Associates, San Antonio, TX, USA), Dr Marcus Hompesch (Profil Institute for Clinical Research Inc., San Diego, CA, USA), Dr Jon Ruckle [Covance (formerly

Radiant), Honolulu, HI, USA] and Dr Royce Morrison [Charles River (formerly Northwest Kinetics), Tacoma, WA, USA].

Samuel C. Ramage assisted in the preparation of this manuscript.

Parts of this study were presented at the 69th Scientific Sessions of the American Diabetes Association, New Orleans, LA, USA, 5 – 9 June 2009 and the 45th Annual Meeting of the European Association for the Study of Diabetes, Vienna, Austria, 29 September to 2 October 2009.

Conflict of Interest All authors contributed to the design, conduct, analysis and manuscript writing. There is no competing interest for any of the authors.

References 1. Kieffer TJ, McIntosh CH, Pederson RA. Degradation of glucose-dependent

insulinotropic polypeptide and truncated glucagon-like peptide 1 in vitro and in vivo by dipeptidyl peptidase IV. Endocrinology 1995; 136: 3585 – 3596.

2. Fehse F, Trautmann M, Holst JJ et al. Exenatide augments first- and second- phase insulin secretion in response to intravenous glucose in subjects with type 2 diabetes. J Clin Endocrinol Metab 2005; 90: 5991 – 5997.

3. Gentilella R, Bianchi C, Rossi A, Rotella CM. Exenatide: a review from pharmacology to clinical practice. Diabetes Obes Metab 2009; 11: 544 – 556.

4. Simonsen L, Holst JJ, Deacon CF. Exendin-4, but not glucagon-like peptide- 1, is cleared exclusively by glomerular filtration in anaesthetised pigs. Diabetologia 2006; 49: 706 – 712.

5. Drucker DJ, Buse JB, Taylor K et al. Exenatide once weekly versus twice daily for the treatment of type 2 diabetes: a randomized, open-label, non-inferiority study. Lancet 2008; 372: 1240 – 1250.

6. Nauck MA, Ratner RE, Kapitza C, Berria R, Boldrin M, Balena R. Treatment with the human once-weekly glucagon-like peptide-1 analog taspoglutide in combination with metformin improves glycaemic control and lowers body weight in patients with type 2 diabetes inadequately controlled with metformin alone: a double-blind placebo-controlled study. Diabetes Care 2009; 32: 1237 – 1243.

7. Vilsboll T, Zdravkovic M, Le-Thi T et al. Liraglutide, a long-acting human glucagon-like peptide-1 analog, given as monotherapy significantly improves glycaemic control and lowers body weight without risk of hypoglycaemia in patients with type 2 diabetes. Diabetes Care 2007; 30: 1608 – 1610.

8. Glaesner W, Vick AM, Millican R et al. Engineering and characterization of the long-acting glucagon-like peptide-1 analogue LY2189265, an Fc fusion protein. Diabetes Metab Res Rev 2010; 26: 287 – 296.

9. Barrington P, Chien JY, Tibaldi F, Showalter HDH, Schneck K, Ellis B. LY2189265, a long-acting glucagon-like peptide-1 analogue, showed a dose-dependent effect on insulin secretion in healthy subjects. Diabetes Obes Metab 2011; 13: 434 – 438.

10. Faries D. Practical modifications of the continual reassessment method for phase I cancer clinical trials. J Biopharm Stat 1994; 4: 147 – 164.

11. Dmitrienko A, Smith B. Repeated-measures models in the analysis of QT intervals. Pharm Stat 2003; 2: 175 – 190.

12. Iltz JL, Baker DE, Setter SM, Keith CR. Exenatide: an incretin mimetic for the treatment of type 2 diabetes mellitus. Clin Ther 2006; 28: 652 – 665.

13. Garber A, Henry R, Ratner R et al. Liraglutide versus glimepiride monother- apy for type 2 diabetes (LEAD-3 Mono): a randomized, 52-week, phase III, double-blind, parallel-treatment trial. Lancet 2009; 373: 473 – 481.

432 Barrington et al. Volume 13 No. 5 May 2011

DIABETES, OBESITY AND METABOLISM original article 14. Umpierrez GE, Blevins T, Rosenstock J, Cheng C, Anderson JH, Bastyr EJ

III, for the EGO Study Group. The effects of LY2189265, a long-acting glucagon-like peptide-1 analogue, in a randomized, placebo-controlled, double-blind study of overweight/obese patients with type 2 diabetes: the EGO study. Diabetes Obes Metab 2011; 13: 418 – 425.

15. Schmidt WE, Siegel EG, Creutzfeldt W. Glucagon-like peptide-1 but not glucagon-like peptide-2 stimulates insulin release from isolated rat pancreatic islets. Diabetologia 1985; 28: 704 – 707.

16. Rickels MR, Schutta MH, Mueller R et al. Islet cell hormonal responses to hypoglycaemia after human islet transplantation for type 1 diabetes. Diabetes 2005; 54: 3205 – 3211.

17. Reaven GM, Chen YD, Golay A, Swislocki AL, Jaspan JB. Documentation of hyperglucagonaemia throughout the day in nonobese and obese patients with noninsulin-dependent diabetes mellitus. J Clin Endocrinol Metab 1987; 64: 106 – 110.

18. Schirra J, Nicolaus M, Roggel R et al. Endogenous glucagon-like peptide 1 controls endocrine pancreatic secretion and antro-pyloro-duodenal motility in humans. Gut 2006; 55: 243 – 251.

19. Meier JJ, Gallwitz B, Schmidt WE, Nauck MA. Glucagon-like peptide 1 as a regulator of food intake and body weight: therapeutic perspectives. Eur J Pharmacol 2002; 440: 269 – 279.

Appendix

Table A1. Test meal glucose AUC relative to placebo after 5 weeks of LY2189265 dosing.

Comparison to placebo

Treatment (n) Week 5 gAUC estimate (mg × h/dl) Difference from placebo 90% CI p-value Placebo (22) 880.45 — — — 0.05 mg LY2189265 (4) 729.89 −150.56 −282.38, −18.74 0.06 0.3 mg LY2189265 (12) 782.46 −97.99 −190.74, −5.24 0.08 1 mg LY2189265 (10) 575.04 −305.42 −401.84, −208.99 <0.01 3 mg LY2189265 (4) 594.14 −286.32 −420.39, −152.24 <0.01 5 mg LY2189265 (16) 612.39 −268.07 −352.59, −183.54 <0.01 8 mg LY2189265 (8) 469.05 −411.40 −511.23, −311.58 <0.01 LSM differences of LY relative to placebo in the change from baseline in gAUC values following the day 36 test meal are shown. For gAUC, ‘n’ is the number of observations from all patients in a treatment group. AUC, area under the curve; LSM, least squares mean.

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Insulin Mean (SD) C-peptide Mean (SD)

Figure A1. Mean area under the curve (AUC) of insulin (shaded box) and C-peptide AUC (striped box) values at each dose are shown normalized to gAUC. Values for insulin AUC normalized to gAUC and C-peptide AUC normalized to gAUC are shown on the left and right axes, respectively. Asterisks indicate statistically significant (p < 0.01) differences from placebo.

Table A2. Test meal glucagon relative to placebo after 5 weeks of dosing.

Comparison to placebo

Treatment (n) LS mean glucagon (pg/ml) Difference from placebo 90% CI p-value

Placebo (11) 5.20 — — — 0.05 mg LY2189265 (2) −2.12 −7.32 −27.84, 13.20 0.55 0.3 mg LY2189265 (6) 3.16 −2.03 −17.58, 13.51 0.83 1 mg LY2189265 (5) 1.54 −3.65 −18.09, 10.78 0.67 3 mg LY2189265 (2) −1.38 −6.58 −27.38, 14.23 0.60 5 mg LY2189265 (8) 8.52 3.32 −9.43, 16.08 0.66 8 mg LY2189265 (4) 7.74 2.55 −14.45, 19.54 0.80 LSM differences of LY relative to placebo in the change from baseline in glucagon values following the day 36 test meal are shown. For glucagon, ‘n’ is the number of observations from all patients in a treatment group. LSM, least squares mean.

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