Pharmacokinetics, Pharmacodynamics, Safety, and Tolerability of a Single-Dose of NN2211, a Long-Acting Glucagon-Like Peptide 1 Derivative, in Healthy Male Subjects Free

The protocol was approved by the Independent Ethics Committee in Manchester, U.K., and conducted in accordance with the Helsinki Declaration and Good Clinical Practice. Subjects consented to participating in the study in writing after a full explanation of the study had been given.

Subjects were recruited from a volunteer database kept at Medeval Ltd. A total of 72 healthy male subjects in good general health, as verified by medical history, physical examination, clinical chemistry, hematology, and electrocardiogram (ECG), aged 18–45 years with BMI 20–27 kg/m², were included in this study. Mean age was 27 years (range 19–41) and weight 74.5 kg (range 58.0–98.0).

This was a single-center, randomized, double-blind, placebo-controlled, single-dose, sequential-dose level escalation study. Eight different dose levels of NN2211 were studied: 1.25 (performed twice), 2.5, 5.0, 10.0, 12.5, 15.0, 17.5, and 20.0 μg/kg with eight subjects at each dose level, and a 3:1 active:placebo randomization within each dose level. Subjects were only enrolled at one dose level. In addition, at each dose level, three subjects were randomly assigned (one placebo/two active) to receive a 2-h intravenous glucose tolerance test (IVGTT). Finally, to determine the absolute bioavailability of NN2211, all eight subjects allocated at the 5 μg/kg subcutaneous dose level returned after a 7-day washout to receive an intravenous dose of 5 μg/kg NN2211 or placebo.

NN2211 at a concentration of 5 mg/ml or matching placebo, manufactured in accordance with good manufacturing practice, was provided in cartridges. These cartridges fit into the NovoPen 1.5, which was used for subcutaneous dose administration. The medication was labeled in accordance with the blinded status of the study. For the intravenous administration of NN2211/placebo performed at the 5 μg/kg dose level to determine the absolute bioavailability, a dilution of the trial medication was performed corresponding to 0.1 mg/ml. The intravenous infusion was performed over 1 h (to avoid high peak plasma concentrations), with an infusion rate calculated based on the weight of the individual subject. Sealed codes were kept at the trial site and at Novo Nordisk in the U.K. and Denmark. A copy of the code was provided only to the responsible person performing the analysis of the concentration of NN2211 in blood and was not further disclosed (because this analysis would unblind the study anyway, it was done to reduce the number of placebo samples analyzed). The code was only broken after database lock but could have been broken for individual subjects in case of an emergency.

The subjects were admitted to the clinic on the day before dose administration and given a standardized meal at ∼7:00 p.m. on the day of admittance and at 6:00 a.m. the following day. NN2211 or placebo was administered between 8:00 and 10:00 a.m. as a single subcutaneous dose in the abdomen. At the first dose level (1.25 μg/kg), the IVGTT was started 5 h postdose; however, because blinded pharmacokinetic data showed that the time to maximal plasma concentration of NN2211 was 9 h in humans, the protocol was amended to start the 2-h IVGTT at 9 h postdose in all subsequent dose levels, including a repeat of the 1.25 μg/kg dose level. A standardized meal was served 11 h after dosing, i.e., after completion of the IVGTT. Blood samples for determination of the concentration of NN2211 were collected at regular intervals from before dosing to 48 h after dosing. Furthermore, a predose to 24-h postdose glucose, insulin, and glucagon profile was obtained in all subjects. In subjects randomized to undergo the IVGTT, additional blood samples for a 2-h glucose, insulin, and glucagon profile were drawn, i.e., between 9 and 11 h after dosing. All subjects were monitored for safety during the study. Adverse events either observed by the investigator or reported spontaneously by the subjects were recorded. In addition, subjects were asked at predose and at 1, 2, 4, 6, 8, 10, 12, 14, 16, 24, and 48 h postdose if they had had any adverse events (including any changes in concomitant illness or new illnesses) since the last evaluation. Clinical laboratory tests (hematology, biochemistry, and urinalysis) were carried out before, during, and after dosing. Blood glucose was measured at regular intervals after dose administration and before discharge using a HemoCue device. Physical examination, ECG (before dosing and 4, 8, 12, 16, 24, and 36 h after dosing), and vital signs, including blood pressure, pulse, and temperature, were recorded. Urine volumes were assessed for the period 0–24 h after dose administration. At the 5-μg/kg dose level, the subjects returned after 1 week for the intravenous administration of NN2211/placebo. At this visit, all procedures were performed as described above, with the exception of the IVGTT, which was not performed.

The concentration of NN2211 was determined by a validated two-site immunoassay using a capturing antibody (GLPB1F1) and a detection antibody (biotin-labeled Mab26.1) as described previously (21). The selectivity of the assay toward NN2211 compared with endogenous GLP-1 was ensured by removing endogenous GLP-1 activity by incubating the samples for 4 h at 37°C before performing the assay. In addition, the interference from a number of peptides was investigated. NN2211 (7-37) isomer interfered positively but <4%, NN2211 (9-37) isomer had negative interference <12%, GLP-1 (15-37) had negative interference <3%, and major pro-glucagon fragment had negative interference <4%. Because all of these interferences were low, and the concentrations of the peptides in the majority of the samples were low compared with the NN2211 concentrations, interference was considered irrelevant. Serum glucose was measured on a Beckman CX5 Delta analyzer using the glucose oxidase technique. Serum insulin was analyzed using the Abbott IMx assay, which shows <0.005% cross-reactivity with human pro-insulin, and glucagon was analyzed with Linco’s glucagon radioimmunoassay in accordance with the manufacturer’s instructions.

Because of the exploratory status of this study (first administration to humans), the sample size of eight subjects per dose level was not based on formal statistical considerations but was considered sufficient to meet the primary objectives of the study. A significance level of 5% was used. Results from the statistical analysis are presented with the mean and 95% CI.

Urine volume (0–24 h) was analyzed by ANOVA with the following model: the response for each subject was the sum of an overall mean, a fixed dose effect (placebo = zero dose), a random block effect of the dose level groups (including the two placebo subjects), and a random error. All 72 subjects enrolled were included.

The pharmacokinetic parameters were assessed by noncompartmental methods from the 48-h NN2211 plasma concentration time profile for the single-dose subcutaneous and intravenous administration. Dose proportionality for subcutaneous dosing was assessed for area under the curve (AUC) to infinity and maximal plasma concentration (C_max) by performing linear regression analysis on the log-transformed parameters versus log-transformed dose. An estimate of the slope of the regression line and corresponding 95% CI was calculated. Dose proportionality was assumed if the slope was not statistically significantly different from unity. Of the 54 subjects exposed to active treatment, 3 and 2 subjects were excluded at the 1.25-μg/kg (first cohort only) and 5-μg/kg dose levels, respectively. This exclusion was due to dosing problems (low dosing volume) that caused low exposure when compared with other subjects at the same dose level.

Only pharmacodynamic data after subcutaneous administration were included in the statistical analysis. The AUC_{0–9 h} (all subjects) and AUC_{9–10 h} and AUC_{9–11 h} (IVGTT-treated subjects only) for glucose, glucagon, and insulin were derived from the respective profiles, applying the trapezoidal rule, and log-transformed. The glucose disappearance constant (K_g) was calculated by linear regression analysis of natural log-transformed glucose data from the IVGTT between 10 and 60 min after glucose administration. For each individual subject, the K_g was defined as the negative slope of the regression line. In one subject receiving placebo, the glucose concentration did not peak until 15 min, and the 10-min data point was therefore omitted for that subject. In another subject receiving placebo, the glucose concentration had rebounded markedly at the 60-min time point, which was therefore omitted for that subject. Exclusion of these data points represents a conservative approach for the assessment of a possible positive effect of NN2211 on K_g. In the statistical model for end points, the response for each subject was the sum of an overall mean, a fixed-dose effect (placebo = zero dose), a random block effect of the dose level groups (including the one or two placebo subjects), and a random error, and the data were analyzed using ANOVA. All subjects from the first cohort of 1.25 μg/kg were excluded because of the timing of the IVGTT. For the AUC_{0–9 h} glucose, insulin, and glucagon analysis, two subjects at 5.0 μg/kg were excluded because of low exposure, and one additional subject was excluded because he did not fast (randomized to placebo at the 17.5-μg/kg dose level), giving a total population of 61 subjects. Because no comparison to placebo could be made, all subjects receiving an IVGTT at the 17.5-μg/kg dose level were additionally excluded in the AUC_{9–11 h} analysis, giving a total population of 20 subjects.

No serious adverse events were reported, and all subjects completed the study. There was a higher number of adverse events reported in subjects on active treatment versus placebo treatment. The higher frequency was noted for headache, dizziness, nausea, and vomiting. Whereas headache and dizziness occurred on the majority of dose levels, nausea and vomiting mainly occurred from 10 and 15 μg/kg, respectively. At the highest dose level, 6 of 6 subjects experienced nausea and/or vomiting compared with 2 subjects experiencing nausea and 1 experiencing vomiting out of 18 subjects in the placebo-treated group. There were no clinically significant changes in vital signs, ECG parameters, physical examination, or safety laboratory parameters (hematology, biochemistry, and urinalysis). There was a tendency toward lower urine volumes at doses ≥12.5 μg/kg, but there was no overall significant difference in urine volumes 0–24 h after dose administration between active and placebo treatment (mean −167; 95% CI [−650 to 316]).

Figure 1A shows the mean plasma concentration time profiles of NN2211 after subcutaneous administration, and the derived pharmacokinetic parameters are given in Table 1. NN2211 was slowly absorbed after subcutaneous administration, with maximum plasma concentrations obtained ∼9–12 h after dosing and a subsequent mean elimination half-life in the range of 11–15 h. After intravenous administration, NN2211 was shown to have a low volume of distribution (0.07 l/kg) and a mean elimination half-life of 8.1 h. The absolute bioavailability based on the intravenous and subcutaneous crossover administration of 5.0 μg/kg was found to be 55%. Figure 1B shows a plot of AUC of NN2211 versus dose, indicating a linear relationship. However, statistical analysis showed linearity only in the dose range of 2.5–20 μg/kg for both C_max (mean slope 1.07 [95% CI 0.91–1.24]) and AUC (1.09 [0.94–1.24]) but not in the range of 1.25–20 μg/kg (1.28 [1.17–1.40] and 1.26 [1.15–1.37] for C_max and AUC, respectively).

The mean 24-h insulin, glucose, and glucagon time profiles are shown in Figs. 2A, 3A, and 4A, respectively. Furthermore, the derived AUC_{0–9 h}/9 h is shown in Table 2. Overall, in the period before the first meal and the IVGTT corresponding to AUC_{0–9 h}, there was a borderline-significant lowering of glucose levels (P = 0.066; mean 0.97 [95% CI 0.94–1.00]) but no statistically significant difference between active and placebo treatment for insulin (0.97 [0.81–1.16]) or glucagon (0.98 [0.91–1.05]). Furthermore, in connection with the meal (corresponding to 11-h postdose) and at doses >10.0 μg/kg, there was a tendency toward lower insulin and glucose levels after active treatment. Concentration time profiles for insulin, glucose, and glucagon during the IVGTT (9–11 h after dose) are shown in Figs. 2B, 3B, and 4B, respectively. Furthermore, the derived AUC_{9–10 h}/1 h and AUC_{9–11 h}/2 h are shown in Table 2, i.e., corresponding to the first hour of the IVGTT and the complete 2-h IVGTT. Although there was a tendency toward higher glucose levels within the first 5 min after the initiation of the IVGTT in the actively treated subjects, a lowering of glucose occurred from 30 to 120 min in the higher-dose groups. There was a clear dose-dependent increase in insulin secretion during the IVGTT, but there was no effect on glucagon levels. Statistical analysis showed a significant increase in insulin levels after NN2211 administration during the IVGTT (AUC_{9–11 h}) (P = 0.0002; mean 2.29 [95% CI 1.80–2.92]) but no significant effect on glucose (0.97 [0.82–1.14]) or glucagon (1.13 [0.89–1.42]). Statistical analysis of the AUC_{9–10 h} during the IVGTT showed a similar pattern to that for the complete IVGTT. Thus, there was a statistically significant increase in insulin levels in the NN2211-treated group compared with the placebo group (P = 0.0002; 2.74 [1.75–4.28]), whereas no significant effect on glucose (1.05 [0.89–1.23]) or glucagon (1.06 [0.87–1.29]) was observed. The effect of NN2211 on intravenous glucose tolerance, as assessed by K_g up to 1 h after glucose administration, is shown in Table 2. There was a clear and significant dose-response relationship (P = 0.0078; slope estimate 0.0713 ± 0.0238 [mean ± SE]), such that increasing doses of NN2211 increased the K_g value. However, the statistical analysis did not show a significant overall difference between NN2211 and placebo treatment (P = 0.16; 0.58 [−0.26 to 1.42]), indicating that NN2211 had an effect on K_g at higher, but not at lower, doses.

The observations in this study of an increase in gastrointestinal side effects and dizziness after NN2211 administration are in accordance with previously published observations after both GLP-1 (7-36) amide infusion and NN2211 administration to healthy male subjects and patients with type 2 diabetes (3,9,21,22). The mechanism behind the occurrence of nausea and vomiting is most likely mediated via inhibition of gastric emptying (11,12,23,24).

Native GLP-1 is a polypeptide that undergoes rapid metabolism by DPP-IV and, in addition, is rapidly cleared by renal elimination (1). The half-life after intravenous or subcutaneous administration in humans has been reported to be ∼5 min (1) and 1 h (2), respectively. Findings in this study show an elimination half-life of 8.1 h after intravenous administration of NN2211. This increase in the half-life of NN2211 compared with native GLP-1 is most likely mediated via a combination of factors. Notably, NN2211 has been shown to have a lower susceptibility to metabolism by DPP-IV (25) and a high degree of albumin binding of NN2211 (as has been shown for other fatty acid derivatives [14,15]), and after subcutaneous administration, an additional prolongation of the half-life is mediated by a slow absorption of NN2211 from the injection site, as evidenced by the further increase in half-life observed from subcutaneous to intravenous administration. Thus, the pharmacokinetic terminal half-life of NN2211 after the subcutaneous administration observed in this study is in accordance with previous findings in pigs, where the half-life was found to be 14 h (17), and studies with NN2211 in humans (21,22) and strongly supports once-daily administration of NN2211. In addition to improvements in compliance and convenience to the patients by the once-daily formulation, an increase in exposure from 16 to 24 h of native GLP-1 showed further improvements in glycemic control, indicating the therapeutic importance of full 24-h GLP-1 exposure (26).

In the present study, a borderline significant lowering in 0–9 h glucose levels after NN2211 administration was observed, whereas no significant effect on 0–9 h insulin or glucagon levels was found. This assessment period was chosen because the subjects were fasted except for water (before IVGTT and meal). Therefore, the previously reported effects in healthy subjects on inhibition of gastric emptying by GLP-1 causing a lower meal-stimulated insulin secretion outweighing the stimulation on insulin secretion caused by GLP-1 would not be present (27). This blunting effect was observed in this study in connection with the meals at ∼11 h after dosing. Previous studies with GLP-1 infusion in healthy subjects have shown an acute effect on insulin and glucose during fasting conditions (4,5), whereas the present study only demonstrated a borderline significant effect on glucose. The basis for this difference remains speculative but could relate to differences in administration form, notably intravenous versus subcutaneous, or sampling frequency differences. However, in accordance with previous findings in both animal models and humans (28), significant increases in insulin levels were observed during the IVGTT in this study. Even though the initial higher glucose values observed during the IVGTT, for which the explanation is not known, could have confounded the observed stimulation of insulin secretion, the effect on insulin was clearly dose related (in contrast to the glucose increase). Although the increased insulin secretion mediated by NN2211 did not significantly decrease glucose levels, there was a dose-dependent increase in the glucose disappearance constant after NN2211 administration, indicating that the lack of a measurable effect on glucose was not caused by NN2211 decreasing insulin sensitivity. In a recent study with NN2211 in patients with type 2 diabetes, NN2211 was found to reduce fasting and meal-related glycemia. This effect was found to be mediated via an increase in insulin secretion rate, suppression of glucagon secretion, and a delay in gastric emptying (22). Thus, in contrast to the present study, suppression of glucagon secretion was found in type 2 diabetic patients after NN2211 administration. Taken together, these data indicate that the pharmacodynamic response of NN2211 in patients with type 2 diabetes is more pronounced that that in healthy subjects and also corroborates the glucose-dependent mechanism of action of GLP-1 and NN2211.

Parts of this study were presented in abstract form at the American Diabetes Association Annual Meeting and Scientific Sessions, Philadelphia, Pennsylvania, 22–26 June 2001.