Pharmacokinetics and Pharmacodynamics of Insulin Glargine Given in the Evening as Compared With in the Morning in Type 2 Diabetes

The insulin dynamic of the postabsorptive and interprandial state of nondiabetic subjects (basal insulin) is best reproduced in people with diabetes requiring insulin administration by the long-acting insulin analogs compared with NPH insulin (1). This more physiological substitution translates into clinical benefits compared with NPH, primarily a reduced risk of nocturnal hypoglycemia for similar A1C levels (1–7).

NPH insulin is most often injected at bedtime to limit the risk of nocturnal hypoglycemia due to its peak of action 4–6 h postinjection (1–3). In contrast, the smoother activity of the long-acting insulin analog glargine allows more flexibility in dosing, and its administration is less strictly bound to the time of injection. Hence, insulin glargine is approved for administration at any time of day, provided it is at the same time each day (8).

However, only a few studies have compared the effects of glargine given in the evening compared with other times of day both in type 1 diabetes mellitus (9–11) as well as in type 2 diabetes mellitus (T2DM) (12,13), with results basically consistent in the noninferiority of morning versus evening dosing. Yet, the large majority of people with diabetes inject glargine in the evening, often at bedtime, in keeping with the tradition of NPH administration.

One reason why the potentially greater flexibility of glargine in the treatment of T2DM has not translated into a recognized option of administration at a different time of day compared with NPH, might be the lack of head-to-head studies of pharmacokinetics (PK) and pharmacodynamics (PD), comparing glargine given in the evening versus the morning. Yet, an indirect comparison of PK and PD between studies, where glargine was given in the evening (14) compared with the morning (15–17), suggests important differences in action profiles, although in such a comparison the contribution of circadian changes in insulin sensitivity should be taken into account (18,19).

The aim of the current study was to perform a head-to-head comparison of the PK and PD of insulin glargine in persons with T2DM, after either evening or morning administration.

After approval by the local ethics committee and after written informed consent was obtained, 10 T2DM persons receiving insulin therapy (all glargine as basal with/without mealtime rapid-acting insulin and oral hypoglycemic agents) were recruited (Table 1) and studied according to the guidelines of the Helsinki Declaration and good clinical practice requirements. Subjects were free of any detectable microangiopathic and macroangiopathic complications and of any major illness other than diabetes, as indicated by medical history, physical examination, electrocardiogram, or routine laboratory tests. All of the subjects were receiving insulin glargine with the evening meal.

The study has a randomized, single-dose, open-label, crossover design (Supplementary Fig. 1). After a 2-week run-in period, during which subjects with diabetes continued their treatment, and the dose of basal insulin glargine was optimized to achieve a fasting plasma glucose (PG) concentration of ≤100 mg/dL while avoiding nocturnal hypoglycemia (PG <72 mg/dL), subjects were randomized to two 24-h euglycemic clamp studies, after either evening (2200 h) or morning (1000 h) subcutaneous injection of insulin glargine (0.4 units/kg). Each occasion was separated by an interval of 3–4 weeks, during which the usual insulin regimen of the run-in period was continued (glargine at dinnertime).

After randomization, subjects were switched in a timely manner from dinnertime to either morning (1000 h, N = 5) or evening (2200 h, N = 5) glargine administration 9 days (day −9) prior to the study day (day 0). For morning dosing, on day −9, subjects injected 50% of the usual glargine dose at dinnertime, followed by the remaining 50% the next morning at 1000 h (day −8). On day −7 and thereafter until the study day, the full glargine dose was injected at 1000 h every day. For evening dosing, subjects injected glargine at 2200 h every day from day −9 until the study day. With either morning or evening dosing, basal insulin concentration was titrated as in the run-in period, and treatment with mealtime rapid-acting insulin was continued, if patients had been receiving such treatment.

On both evening and morning study occasions, subjects had the last subcutaneous glargine injection 24 h before the time they received glargine on the study day. An equivalent interval of fasting was observed before initiation of each clamp study (14 h), with the last meal (688 kcal, 54% carbohydrate, 30% protein, and 16% lipids) consumed either at 0800 h the same day of the study (glargine evening) or at 2000 h the day before the study (glargine morning). Care was taken by subjects to avoid hypoglycemia (PG <72 mg/dL) with blood glucose self-monitoring in the 48 h before the clamp studies. For the entire duration of the studies, all persons monitored blood glucose levels by means of a reflectometer (OneTouch Vita; LifeScan Italy [part of Johnson & Johnson Medical S.p.A.], Milan, Italy).

Persons with diabetes were admitted in the fasting state to the Clinical Research Unit of the Department of Medicine, Section of Internal Medicine, Endocrinology and Metabolism of the University of Perugia Medical School, 4 h prior to study initiation (i.e., at either 0600 or 1800 h, respectively, for the morning and evening studies), and remained fasting for the subsequent 28 h (Supplementary Fig. 1). They were put at rest in a bed and studied with the euglycemic clamp technique (20). In brief, two venous lines were inserted, an antecubital vein on one arm (for infusions) and a dorsal vein (cannulated retrogradely with a 20-gauge butterfly needle) on the contralateral hand (kept at 65°C in a hot box) for intermittent sampling of arterialized venous blood. An intravenous feedback insulin infusion was initiated to achieve and maintain PG at 100 ± 5 mg/dL until time 0 (i.e., 1000 or 2200 h), as previously described (21). At −120 min, a primed sterile, pyrogen-free constant infusion (0.222 μmol/kg/min) of [6,6-²H₂]-glucose (Cambridge Isotope Laboratories, Cambridge, MA) was started and maintained throughout the experiment to determine glucose kinetics. A variable rate of a 20% dextrose solution enriched to 4% with [6,6-²H₂]-glucose was used to clamp the PG concentration at the desired target of 100 mg/dL for 24 h (14) and to avoid non–steady-state errors in the measurement of glucose turnover, as described previously (22). At 1000 or 2200 h, a subcutaneous injection of 0.4 units/kg insulin glargine (Lantus 100 units/mL) was administered by means of injection with a 0.5-mL insulin syringe into the abdominal area. On both occasions, the clamp procedure was terminated at 24 h after the subcutaneous injection of glargine.

PG concentration was measured at the bedside using the YSI 2300 glucose analyzer (YSI 2300 STAT; Yellow Springs Instruments, Yellow Springs, OH). The plasma free insulin concentration was measured by radioimmunoassay after polyethylene glycol extraction of antibodies from the plasma (23). Plasma free fatty acid (FFA), glycerol, β-OH-butyrate, C-peptide, and glucagon concentrations were measured by previously described methods (14). Glucose enrichment was determined on its penta-acetate (penta-O-acetyl-β-d-glucopyranose) derivative by gas chromatography–mass spectrometry (HP 6890 II GC, HP 5973A GC/MS; Hewlett-Packard, Palo Alto, CA) in electron impact ionization mode monitoring the ions 200 and 202 for the unlabeled and [6,6-²H₂]-glucose, respectively (24).

Calculations of glucose fluxes are based on a non–steady-state assumption and the total R_a and R_d values were calculated using a modified form of the Steele equation to account for the addition of stable labeled tracer to the exogenous glucose infusate (24). Endogenous glucose production (EGP) was obtained as the difference between R_a and the exogenous glucose infusion rate (GIR) during the clamp. Because the isotopic steady state might have not been achieved during the first hours of the study (25), in order to have reliable values of glucose fluxes, calculations were performed by excluding the first 6 h of the study.

The linear trapezoidal rule was used to calculate the concentration-time area under the curve (AUC) between 0 and 24 h (AUC_0–24h) for GIRs, plasma free insulin, and nonglucose substrates. The maximum plasma concentration (C_max) and the time to reach C_max (T_max) for the same variables were read directly from the plasma concentration-time data for each subject. The determinations of C_max and T_max for the GIR were derived from a smoothed 3-point running average GIR curve for each subject in order to provide reliable data for calculation.

The primary analysis of the PK and PD parameters was performed using ANOVA, which allowed for variation of subjects nested within sequence, period, and treatment. T_max variables were analyzed nonparametrically (Wilcoxon rank sum test and Hodges-Lehmann estimates of the treatment with 95% confidence limits).

The primary end point of the study was the GIR over the first 12 h (AUC_0–12h) and the GIR over the second 12 h (AUC_12–24h). Secondary end points were PG concentration; glucose fluxes (AUC_7–24h); plasma C-peptide, glucagon, and nonglucose substrate concentrations; intravenous insulin infusion rates prior to and immediately after subcutaneous glargine insulin injection; and plasma insulin concentrations. A sample size of 10 subjects was chosen in order to achieve 80% power to detect a mean of paired differences of 40% with an estimated SD of differences of 40% between the GIR for glargine infused during both the first 12 h (AUC_0–12h for glargine at 1000 h vs. glargine at 2200 h) and the second 12 h (AUC_12–24h for glargine at 1000 h vs. glargine at 2200 h). All tests of statistical hypothesis were carried out at the 5% level of significance and comparisons were two sided. Data in the text are expressed as the mean ± SD and median with 25th and 75th percentiles as appropriate, and in figures as the mean ± SE. Statistical analysis was usually performed using PASS (NCSS Statistical Software, Kaysville, UT).

All 10 subjects enrolled completed the two studies.

After randomization, home daily glycemic control (mean PG from self-monitoring data) during the 7 days before the clamp studies was not different in the morning study (139 ± 11 mg/dL) versus the evening study (142 ± 7 mg/dL, P = 0.221). The dose of glargine over the 7 days prior to the studies was slightly, but significantly, higher in the morning versus the evening study (0.31 ± 0.03 vs. 0.29 ± 0.03 units/kg, P = 0.002). Also, total daily insulin doses were greater in the morning (0.56 ± 0.3 units/kg) compared with the evening study (0.55 ± 0.3 units/kg), although the difference did not reach statistical significance (P = 0.070).

The amount of insulin infused to achieve and maintain euglycemia during the feedback period (time −2 to 2 h before and after time 0 h) was greater with glargine given in the morning (0.3 ± 0.2 mU/kg/min) compared with the evening (0.2 ± 0.2 mU/kg/min), but the difference did not reach statistical significance (P = 0.190). After the subcutaneous injection of glargine at time 0 h, the rate of intravenous insulin infusion decreased in both treatments. However, the withdrawal of intravenous insulin infusion tended to be faster with evening (time −1 ± 1 h) compared with morning glargine dosing (time 0.3 ± 1.3 h) (P < 0.065) (Fig. 1 and Table 2).

At time 0 min, the plasma insulin concentration tended to be higher in the morning versus the evening study (P = 0.082). The overall plasma free insulin concentration after subcutaneous glargine injection did not differ between the two treatments (Fig. 1 and Table 2).

PG concentration decreased similarly during intravenous insulin infusion prior to glargine injection on the two occasions (data not shown), and at time 0 h it was not different in the two studies (102 ± 4.3 and 100 ± 2.2 mg/dL, respectively, for morning vs. evening study, P = 0.061). After glargine injection, the mean PG over the 24-h study period was superimposable in the morning and evening studies. During the first 12 h of the study, the two treatment groups had similar mean PG concentrations, in the second half of the study period (time 12–24 h), the mean PG concentration was slightly, but significantly, higher with morning compared with evening glargine dosing. The PG concentration at the end of the clamp was higher with morning glargine compared with evening injection (108 ± 8 vs. 100 ± 2 mg/dL, P = 0.033) (Fig. 1).

The GIR needed to maintain euglycemia was significantly different between the two studies at time 0 h: positive (0.45 ± 0.55 mg/kg/min) after the evening compared with morning dosing, where it was basically turned off (P = 0.038). The mean GIR for the 24-h study period (AUC_0–24h) was equivalent in the two studies. However, over the initial 12-h period, the GIR was greater in the morning compared with evening study, whereas the opposite was observed for the second 12-h period. Although the maximum GIR values were similar with both treatments, the GIR T_max occurred earlier after morning injection than after evening dosing (Fig. 1 and Table 2).

EGP AUC_7–24h, calculated during the last 17 h of the study at isotopic steady state, was not different with evening compared with morning dosing. However, the EGP exhibited opposite profiles between day and night, indicating greater insulin sensitivity during the day compared with the night. In fact, EGP decreased from a peak value of 1.14 ± 0.57 mg/kg/min at 0800 h to the lowest value of 0.14 ± 0.18 mg/kg/min at 1800 h (evening insulin injection), whereas it increased steadily from 2200 h (0.20 ± 0.21 mg/kg/min) over the night hours to achieve a peak value of 0.96 ± 0.30 mg/kg/min between 0800 and 1000 h (morning insulin injection).

Glucose utilization (GU) was not stimulated and overall was not different among insulin treatments (Fig. 1 and Table 2).

Overall, the plasma C-peptide concentration was similarly suppressed with both treatments (P = 0.104) (Fig. 2 and Table 2).

Plasma glucagon concentrations were more suppressed with evening compared with morning dosing. However, over the initial 12-h period (AUC_0–12h), the degree of suppression was equivalent, whereas in the second half of observation period it was less suppressed with morning glargine dosing (Fig. 2).

Plasma FFA concentrations were more suppressed with evening versus morning glargine dosing over the 24-h study period. During the last 12 h with both treatments, FFA plasma concentrations tended to increase compared with the initial 12 h of the clamp; however, this increase was greater with morning glargine dosing (Fig. 2).

Similarly, plasma β-OH-butyrate concentrations were lower with evening glargine dosing over the whole study period (0–24 h) (P = 0.005). Indeed, the plasma β-OH-butyrate concentration increased to 1.43 ± 0.99 mmol/L by the end of study with the morning dosing, but only to 0.42 ± 0.32 mmol/L with evening glargine dosing (P = 0.003) (Fig. 2).

After initial suppression, plasma glycerol concentrations increased in both treatments, and remained significantly lower after evening compared with morning dosing (P = 0.020) (data not shown).

Lactate and alanine concentrations were not different between treatments (data not shown).

When insulin activity (as indicated by GIR, EGP, and GU profiles) was analyzed by 24-h clock time, comparing daytime (1000–2200 h) versus nighttime (2200–1000 h) periods from the last 12 h of each study, the GIR was greater during the day versus the night independent of the time of glargine injection (AUC_12–24h 701 ± 396 vs. 412 ± 331 mg/kg × 12 h day vs. night, respectively, P = 0.002) (Fig. 3). EGP changes were consistent with differences in GIR requirements (AUC_12–24h 290 ± 204 vs. 412 ± 202 mg/kg × 12 h day vs. night, respectively, P = 0.041) (Fig. 3). GU did not change and was not different between treatments (P = 0.079 and P = 0.406, respectively, for time and treatment × time interaction, from repeated-measures ANOVA) (Fig. 3).

This is the first study directly comparing in T2DM subjects the PK and PD of basal insulin glargine administered at different times of the day (i.e., in the evening and in the morning). The question is relevant not only in light of the potential clinical translation of the findings, but also considering the great emphasis recently given to the concept of flexibility of dosing time, which has been advocated mainly for the second-generation long-acting insulin analogs (26).

The results indicate that overall 24-h insulin activity was not different whether glargine was injected in the evening or in the morning. However, relevant differences emerged when considering time action profiles and distribution of metabolic effect in the first 12-h period compared with the second 12-h period. Glargine administration in the morning exerted greater activity in the first 12 h, in contrast to evening dosing, which exhibited higher potency in the second 12 h. In addition, the latter was more effective in suppressing overall lipolysis and plasma glucagon concentrations, primarily over the last 12 h of the studies.

In the current study, morning glargine administration induced a robust increase in GIR during the first 12 h, whereas in the second 12 h of the study a progressive decline in GIR occurred, as the result of a rebound in EGP, and PG tended to increase (Fig. 1). This action profile is remarkably in line with previous reports (15–17) in which glargine PD was studied after morning injection. In contrast, with evening glargine the distribution of the metabolic effect was quite different. In the first 12 h, GIR was nearly flat, whereas in the second 12 h, GIR increased notably as the result of the suppression of EGP. The superior insulin activity at the end of the 24-h period after evening glargine administration is also indirectly suggested by the lower rate of intravenous insulin infusion and greater GIR required to reach euglycemia prior to clamp initiation before insulin injection at 2200 h, compared with the rates infused before the morning clamp.

None of the two glargine dosing regimens increased glucose disposal. This is in line with the physiological concept that when basal insulin is replaced at therapeutic doses to target euglycemia, it exerts its effects primarily at the liver and adipose tissue level (suppression of glucose production and lipolysis) without promoting muscle glucose uptake (17,27).

Morning and evening glargine administration had differential effects on 24-h lipolysis and plasma glucagon concentrations. The evening glargine administration, more effectively than morning, restrained lipolysis and ketogenesis, and suppressed plasma glucagon concentrations.

It is tempting to speculate about the mechanisms by which glargine when given in the morning has differential metabolic effects compared with evening administration. One explanation might be different PK in the first 12-h period compared with the second 12-h period (Fig. 1). However, plasma insulin concentrations were relatively stable throughout the 24-h study, without any difference between first and second 12-h periods, after both morning and evening dosing. This applies to the suppression of plasma C-peptide as well (Fig. 2).

The alternative hypothesis might be that the results are primarily related to differential responses to insulin at different times of day (i.e., insulin sensitivity might be greater during daytime hours compared with nighttime hours). In fact, when the data are analyzed by 24-h clock time (Fig. 3), it appears that EGP, the driver of basal insulin effects on glucose metabolism (i.e., GIR), is less suppressed during the night (2200–1000 h) than during the day (1000–2200 h), and to a similar extent in morning and evening dosing studies. At the end of the night, at 1000 h, EGP surprisingly reached similar values after both morning and evening dosing. However, the incremental increase in EGP was greater with morning glargine dosing compared with evening glargine dosing, possibly due to more accelerated lipolysis and greater plasma glucagon concentrations. Conversely, during the day (1000–2200 h), insulin activity increases progressively as a result of the suppression of EGP. The latter, again surprisingly, reaches similar values at 2200 h after either morning or evening glargine administration. Taken together, these differences speak in favor of the circadian rhythm of insulin sensitivity, with greater sensitivity during the day compared with the night (19). Thus, the effects of glargine administered in the morning or evening only appear to differ. They are remarkably reproducible when analyzed by absolute time of day, not the relative number of hours postinjection.

The results of the current study closely reproduce those observed by Radziuk and Pye (28), and represent additional evidence of the dawn phenomenon in T2DM (29–31).

The existence of diurnal rhythmicity in insulin sensitivity driving changes in PG concentration in normal subjects and in T2DM patients has been reported by older (32,33) and more recent studies (18,19,34,35). Boden et al. (19) described a robust and well-defined rhythm in insulin sensitivity in T2DM patients, with lower sensitivity in the morning and higher sensitivity in the evening. Similar to the current study, the circadian changes in GIR were completely accounted for by changes in EGP, whereas the R_d did not change (19). The elegant work of Radziuk and Pye (28) provided a deeper and fascinating insight with the hypothesis that the observed diurnal temporal pattern in EGP might result from factors counteracting insulin action and triggered by the brain through central regulatory pathways. Nowadays, the concept of diurnal rhythmicity in insulin sensitivity finds novel relevance in light of the more extensive concept of a “molecular clock,” which has been shown to regulate the expression and function of a variety of enzymes, transport systems, and nuclear receptors involved in lipid and carbohydrate metabolism (36–38).

Regardless of the mechanisms, the differential insulin activity observed in the current study after morning glargine administration compared with evening glargine administration is relevant to clinical practice. Indeed, insulin glargine provides a rather flat activity profile at night whether given in the morning or in the evening. This explains the reported lower risk of nocturnal hypoglycemia with glargine compared with “peak insulin” such as NPH (4–7). On the other hand, the present studies indicate similarly greater insulin activity during the daytime after glargine administration in either the morning or the evening. This may predict potentiation of the risk of hypoglycemia in the afternoon, for example, when prandial glucose-lowering drugs are combined with basal insulin. Whether dosing at a more usual breakfast time (0700–0800 h) would result in circadian PD effects similar to those observed in the current study cannot be directly derived from our results. However, the time curve of GIR indicates lower insulin sensitivity in the interval 0200–1000 h, suggesting that insulin dosing within this time frame would produce lesser effects until 1000 h and greater effects thereafter.

Since the PD results of the present studies observed with glargine might be interpreted not on the basis of PK, but rather on the circadian rhythm of insulin sensitivity, it is likely that similar effects may occur also with other long-acting insulin formulations. In a recent study (14), NPH and detemir exhibited greater activity in the afternoon compared with morning hours after evening injection in T2DM patients. However, a direct comparison with morning and evening dosing of other long-acting insulins (degludec, already on market outside the US, and glargine 300 units as well as pegylated lispro, which has not yet been approved) is needed.

A limitation of our study is the relatively small number of subjects studied. However, the homogeneity of the data observed and the crossover design reinforce the findings.

A strength of our study is its design, which mimicked real-life conditions as closely as possible: an insulin dose close to that used by the insulin-treated subjects was administered, and the study was performed after multiple dosing of glargine (steady state).

Finally, the results of the current study are relevant to the understanding of PD in clamp studies after subcutaneous injection of long-acting insulin analogs in T2DM (39,40). Distribution of the metabolic effect may be quite different if the insulin to be studied is injected in the morning compared with the evening. Therefore, it would not be correct to assume that the greater metabolic activity of glargine, observed 6–8 h after morning dosing (15–17), is representative of the true action profile since in the current study this appears quite different when glargine is administered in the evening.

In conclusion, total insulin activity on glucose metabolism is similar with evening or morning glargine administration. However, with evening glargine administration, the suppression of nocturnal EGP, lipolysis, and glucagon concentration are more consistent. As result, targeting fasting euglycemia appears more convenient with evening glargine dosing compared with morning glargine dosing. Conversely, morning dosing may be preferable whenever greater protection against the risk for nocturnal hypoglycemia is needed.

Acknowledgments. The authors thank Dr. Gianluca Curti, Department of Medicine, University of Perugia Medical School, Perugia, Italy, for his skillful technical assistance in performing the gas chromatography–mass spectrometry assay for glucose kinetics. This article is dedicated to the subjects with type 2 diabetes who have volunteered for the studies.

Duality of Interest. F.P. has received honoraria for speaker fees and/or travel grants from Sanofi, Eli Lilly & Co, Bristol-Myers Squibb, and Merck & Co. P.L. has received travel grants for scientific meetings from Sanofi and Menarini. G.B.B. has received honoraria from Sanofi, MannKind, and Eli Lilly & Co. for scientific advising and consulting. C.G.F. has served on scientific advisory panels for Sanofi and has received honoraria for speaker fees and/or travel grants from Bristol-Myers Squibb, Merck & Co., and Menarini. No other potential conflicts of interest relevant to this article were reported.

Author Contributions. F.P. enrolled patients, performed clamps, analyzed data, and wrote the manuscript. P.L. performed clamps and glucose turnover measurements, analyzed data, and reviewed and edited the manuscript. P.Ci. performed clamps and laboratory assays and reviewed and edited the manuscript. P.Ca., A.M.A., and S.M. performed clamps and reviewed and edited the manuscript. M.A. performed glucose turnover measurements and reviewed and edited the manuscript. G.B.B. provided the study concept and design, supervised the protocol development and the research, contributed to discussion, and reviewed and edited the manuscript. C.G.F. performed clamps, analyzed data, performed the statistical analysis, contributed to discussion, and reviewed and edited the manuscript. F.P. 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. Parts of this study were presented in abstract form at the 73rd Scientific Sessions of the American Diabetes Association, Chicago, IL, 21–25 June 2013.