Euglycaemic glucose clamp: what it can and cannot do, and how to do it

Transcription

1 Received: 14 March 2016 Revised: 5 June 2016 Accepted: 6 June 2016 DOI /dom REVIEW ARTICLE Euglycaemic glucose clamp: what it can and cannot do, and how to do it Tim Heise MD 1 Eric Zijlstra PhD 1 Leszek Nosek MD 1 Sascha Heckermann 1 Leona Plum-Mörschel MD 2 Thomas Forst MD 1,2 1 Profil, Neuss, Germany 2 Profil, Mainz, Germany Conflict of interests: All authors have conducted glucose clamp studies as contract research for pharmaceutical companies. In addition, the authors are employees of and T.H. is part-owner of Profil Germany, the manufacturer and exclusive user of ClampArt, a modern automated glucose clamp device. Corresponding Author: Dr Tim Heise, Profil Institut für Stoffwechselforschung GmbH, Hellersbergstr. 9, D Neuss, Germany (tim.heise@profil.com). The hyperinsulinaemic-euglycaemic glucose clamp has always been regarded as the gold standard for the assessment of pharmacodynamic (PD) properties of insulin preparations; however, there has been controversy over a variety of methodogical details, such as study population, dosing time and the initial stabilization of blood glucose (BG) concentrations at the clamp target level, among clamp groups. As the impact of these details on PD results is unclear, the present review provides an overview of different methodological approaches for both the manual and the automated hyperinsulinaemic-euglycaemic glucose clamp. The advantages and limitations of several methodological details are discussed as well as the relevance of clamp results for the prediction of clinical outcomes. Overall, the best method strongly depends on the exact objective of the trial. If, for instance, duration of action is the primary objective, studies should be carried out in patients with type 1 diabetes to avoid any interference of endogenous insulin. This is less important for variables such as onset of action or early metabolic activity. The hyperinsulinaemic-euglycaemic glucose clamp has a high sensitivity to detect even minor differences between different insulin preparations. The practical relevance of potential differences, however, needs to be investigated in clinical studies. A major prerequisite for obtaining reliable glucose clamp results is the attainment of high clamp quality (i.e. keeping BG concentrations close to the clamp target throughout the experiments). Unfortunately, measures of clamp quality are often under-reported, as is the variability in PD profiles, although these might explain some unconfirmed extreme results obtained in a few clamp studies. KEYWORDS biosimilar insulin, experimental pharmacology, glucose clamp, insulin analogues, pharmacodynamics, pharmacokinetics 1 INTRODUCTION The hyperinsulinaemic-euglycaemic glucose clamp, originally designed for the assessment of insulin sensitivity, quickly became the gold standard for the assessment of pharmacodynamic (PD) characteristics of insulin preparations more than 25 years ago, 1 and regulatory guidelines required glucose clamp results for the development of new insulin preparations. 2 The value of glucose clamps for PD assessments per se had not been questioned until recently, when two commentaries queried the suitability of the glucose clamp technique for the demonstration of bioequivalence between insulin preparations because of alleged methodological shortcomings. 3,4 However, even before this, clamp experts had discussed and sometimes disagreed on methodological details, 5 making it difficult to assess the PD results of glucose clamp studies for non-experts, particularly when these results differed between clamp groups. It might therefore be time to review both the strengths and the limitations of the clamp technique, addressing the most important methodological issues and discussing their potential impact on PD outcomes. 2 GLUCOSE CLAMP STUDIES: PRINCIPLE AND BASIC CONSIDERATIONS The principle of the PD glucose clamp is quite simple 1 : the blood glucose (BG)-lowering effect of an insulin is antagonized by glucose John Wiley & Sons Ltd wileyonlinelibrary.com/journal/dom Diabetes, Obesity and Metabolism 2016; 18(10):

2 HEISE ET AL. 963 infused at a variable rate, so that BG is clamped at a predefined target (the term hyperinsulinaemic is used to indicate that serum insulin concentrations induced by the exogenous are supra-physiological to induce a BG-lowering effect). In manual clamps, the investigator performs BG measurements and manually adjusts glucose infusion rates (GIRs) every 3-10 minutes, whereas in automated clamps, BG is measured continuously and GIR is adapted every minute by an implemented algorithm (and therefore with minimized potential bias). Thus, blinding of investigators is particularly important for manual clamps, although also desirable for automated clamp studies. The performance of automated glucose clamps has for a long time been hampered by the outdated technology of the Biostator, a clamp device developed in the early 1970s. Recently, more modern devices have been introduced: the STG-22/STG-55 (Nikkiso, Tokyo, Japan, only available in Japan) and ClampArt (proprietary to the manufacturer Profil, Neuss, Germany). 6 The different frequencies in BG measurements and GIR adaptations lead to different shapes of the resulting GIR profiles, with many small plateaus occurring with the manual and pronounced oscillations occurring with the automated technique (Figure 1). Nevertheless, if BG can be kept close to target, GIR should reflect the true PD activity, regardless of the clamp technique used. Technically, every deviation from the target means that a wrong GIR was chosen. While short-term and minor deviations from the target are unavoidable, more pronounced or longer-term deviations might mean that the obtained GIRs during these periods do not reflect the true metabolic activity of the investigated insulin. Unfortunately, variables that define glucose clamp quality (i.e. BG deviations from target) have often been under-reported in glucose clamp studies. Some older publications reported the coefficient of variation (%) of BG measurements (which we later named precision ), 6 but this variable alone is not very helpful as it does not provide any information on deviations from the clamp target. We therefore recently introduced additional measures of glucose clamp quality, 6 in particular, the mean difference between measured and target glucose level ( control deviation ). Without information on glucose clamp quality, the validity of the obtained GIR results cannot be properly evaluated, so that measures of clamp quality should be included in every publication on clamp studies, particularly for clamps with rapid-acting or ultra-rapid insulins which because of their fast and pronounced early metabolic effect are more difficult to clamp than slower-acting insulins. 3 GLUCOSE CLAMPS: MOST IMPORTANT OUTCOMES While GIR profiles nicely illustrate the metabolic effect of the investigated insulin over time, GIR-related variables have been developed for statistical comparisons. Of these, total area under the GIR curve (AUC GIR ) and maximum action (GIR max ) are arguably the most important. For prandial insulins, measures of early action are likewise important, in particular, (partial) AUC GIR in the first 1-2 hours after injection, time to maximum action (t-gir max ) and time to early half-maximum action (early t-gir 50% ). 7 For basal insulins, important variables are related to the flatness and even distribution of the metabolic activity profile; for example, the proportion of AUC GIR,0-6 hours, AUC GIR,6-12 hours, AUC GIR,12-18 hours, and AUC GIR,18-24 hours to the total GIR effect over 24 hours (which ideally should be close to 25% for each interval) 8 or the fluctuation of the GIR effect per h [AUC- F GIR,τ = (AUC above GIR mean + AUC below GIR mean )/24 (in a 24-hour interval)]. Usually, GIR profiles are smoothed (in particular with automated clamps) to reduce the impact of artificial GIR oscillations, particularly on time-related parameters (e.g. time to maximum action). While the most frequently used smoothing method is LOESS, a locally weighted regression technique, no real standard has been established yet, and important details such as the extent of smoothing are not always included in publications. It has been rightly pointed out that the extent of smoothing impacts the shape of the obtained GIR profiles and related variables (Figure 2). 8 Common smoothing standards are needed to allow reliable comparisons across different clamp studies or, even better, new algorithms leading to fewer GIR fluctuations and abolishing the need for smoothing. 9 While calculated PD variables are usually reported as mean values, with a variability estimate (standard deviation or standard error) or as least squares means with confidence intervals, PD plots A B FIGURE 1 Differences between the results of manual and automated clamps are visualized in this figure. Individual GIR data of human regular insulin obtained with the manual (A) and the automated glucose clamp technique (Biostator; B) show clear differences in the shape of the profiles with GIR plateaus with the manual and GIR oscillations with the automated technique.

3 964 HEISE ET AL GIR (mg/kg/min) Unsmoothed GIR Smoothing factor 0.1 Smoothing factor 0.25 Time (h) FIGURE 2 Impact of different smoothing procedures on the recorded GIR profiles. The graph shows the first 6 hours of glucose infusion profiles after injection of 0.6 U/kg of Humalog Mix 25/75 (25% insulin lispro, 75% of protaminated insulin lispro). The GIR profile (unsmoothed in black, thin line) was smoothed with LOESS, a locally weighted regression technique, using two different smoothing factors. The smoothing factor gives the percentage of values used for the regression (i.e. a smoothing factor of 0.1 indicates that 10% of values are used for the smoothing). The higher smoothing factor leads to more even profiles (i.e. the GIR oscillations have less impact), but can easily lead to an upward shift of the smoothed profile at the beginning. This artefact leads to elevated GIR of the smoothed profile already at time point 0 (the time of study drug administration) in contrast to the unsmoothed or less smoothed profiles showing a later increase in GIR. (smoothed or unsmoothed) often only show mean values without variation. It has therefore been suggested that all individual PD plots should be shown in addition to mean plots, 5 which has, however, only been implemented in a few publications. 8 While individual plots are certainly better than no variability estimates at all, it is quite difficult to compare inter-individual variability between treatments from several individual plots. Nowadays, it is feasible to show variability bands, even for automated clamps with minute-by-minute GIR values, as shown by some recent (and hopefully many more future) publications. 10,11 4 GLUCOSE CLAMPS: DESIGN ISSUES Overall, the whole set-up of glucose clamp studies aims for a high degree of standardization: study participants are required to fast for at least hours before the start of the clamp, should avoid any strenuous exercise as well as certain foods and drinks in the last days before the clamp, should wash out potentially interfering antidiabetic compounds, in particular insulin, and are required to stay in a supine or semi-supine position and remain fasted throughout the whole experiment. Thus, as for many other pharmaceutical test procedures, the set-up of the PD clamp is aimed at optimizing the sensitivity for identifying potential differences between the investigated insulins rather than at establishing real-life conditions. 3 Sampling for BG concentrations during clamps is usually carried out from arterialized blood using the heated-hand technique. Whether or not this is really necessary remains doubtful. While some studies have shown small differences in BG concentrations between arterial, arterialized, venous and capillary samples, 12 the impact on GIR has been very minor. 13,14 It might therefore be more important to pay attention to other potential confounders, in particular, placement of the blood sampling catheter in a large vein with good blood flow (traditionally retrograde placement has been used in clamp studies, but the importance of this manoeuvre has never been evaluated) and correct pre-analytical handling of the BG samples to avoid false high measurements attributable to haemolysis. Most importantly, laboratory BG analysers with low (<2%) inter- and intra-assay variability should be used. The main focus of PD investigations usually lies in the beginning (onset of action) and the end (duration of action) of clamp experiments. A number of design issues can affect the obtained results, particularly in the areas of interest as described below. 4.1 Study population Major concerns have been expressed with the interpretation of glucose clamp studies in healthy people or people with type 2 diabetes (T2DM) because of the potential interference of endogenous insulin. 3 Indeed, with low pharmacokinetic (PK)/PD effects of an exogenous insulin (mainly basal insulins, particularly at the end of a treatment period), there is a risk that endogenous insulin secretion will be insufficiently suppressed, contributing to the observed GIR effect. Attempts to suppress endogenous insulin secretion by an intravenous insulin infusion starting before dosing and continuing throughout the clamp have not been able to solve this issue, as the effect of this infusion could not be reliably determined in a pre-dosing baseline period of 2 hours. 15 In fact, the GIR effect of a continuous insulin infusion has been shown to increase over at least 4-6 hours. 16 Previous attempts to use somatostatin for the suppression of endogenous insulin have largely been abandoned because of side effects (headache, hypoglycaemia after clamp termination) and potential effects of somatostatin on insulin clearance. Clamp experts therefore agreed already in 2008 that duration of action and late metabolic activity of basal insulins cannot be reliably assessed in healthy people 5 (the early part of the PD profile is usually not too much affected by endogenous insulin secretion) and consequently the European Medicines Agency guideline for biosimilar insulins recommends PK/PD investigations of long-acting insulins in people with type 1 diabetes (T1DM). 17 While endogenous insulin could also be stimulated in T2DM under clamp conditions, similar stimulations might occur under clinical conditions; therefore, it is unclear whether clamp results obtained in T1DM can easily be transferred to T2DM. Small differences between insulins in T1DM might not be of clinical relevance for T2DM, as has been illustrated by differences in the PD profiles of both glargine and detemir between T1DM 18,19 and T2DM. 20 It should be noted, though, that PD clamps in T2DM are usually carried out in insulintreated patients with impaired endogenous insulin secretion as indicated by low fasting C-peptide levels ( 1 nmol/l). 20 While this might minimize the risk of endogenous insulin stimulation, the observed PD results might not be applicable to other people with T2DM, in particular those with high endogenous insulin secretion capacity (see

4 HEISE ET AL. 965 below). Low/negative fasting C-peptide levels should also be proven in people diagnosed with T1DM to exclude residual insulin secretion. 4.2 Stabilization of blood glucose to the clamp level prior to dosing To avoid carry-over effects of the patients usual insulin therapy patients usually only use short-acting insulins in the last 24 hours pre-dosing. While such a regimen prevents metabolic decompensations, it often goes along with short-term BG deteriorations and many patients, in particular those with T1DM, have high BG levels before the glucose clamp. Intravenous insulin (or analogues) is used to stabilize BG at the basal clamp level as it only has a half-life of a few minutes; however, the duration of action is considerably longer, so that concerns have been expressed that the intravenous exogenous insulin bugs the PD data. 4 A carry-over effect can be safely avoided by carefully titrating the intravenous insulin to a rate that keeps BG concentrations constant, but avoids any glucose infusion (Figure 3), so that the observed GIR effect post-dosing is solely attributable to the study insulin. Much experience is needed, however, to achieve really stable BG concentrations prior to dosing with the use of intravenous insulin only. The amount of intravenous insulin needed is quite small. In our experience, 0.5 U intravenous insulin is required in the last hour pre-dosing in T1DM, which argues against a significant impact on PD effects. Potential carry-over effects of intravenous insulin are not a concern in healthy people and less of a concern in patients with T2DM who usually tolerate the withdrawal of previous insulin better and do not show as pronounced BG elevations prior to the clamp as do patients with T1DM. It is therefore possible to investigate insulins with a rather pronounced early metabolic effect in healthy people or those with T2DM, 17 but C-peptide levels should be carefully monitored and experiments with significant rises in C-peptide level from baseline (e.g. >0.2 nmol/l) should be excluded from the analysis. 4.3 Measuring onset of action The assessment of when insulin action starts is of particular importance for fast-acting insulins; however, onset of action is difficult to determine reliably, as BG concentrations below target level early post-dosing do not always indicate onset of action but might occur just because of the variance in BG analysis and physiological BG fluctuations. For basal insulins, it has been proposed to continue intravenous insulin infusion post-dosing with a gradual decrease in the infusion rate until a complete stop. Onset of action was defined as the time when the insulin infusion rate was consistently decreased by 50% compared with the 20-minute pre-injection time period; 21 however, as there are no clear criteria on how to reduce the insulin infusion rate, this definition is prone to substantial bias. Furthermore, the continuation of the intravenous insulin infusion post-dosing might contribute to the observed GIR effect, which is clearly undesired. To overcome these difficulties, we recently modified the glucose clamp procedure for fast-acting insulins and let BG levels drop postdosing before initiating glucose infusion. 7 The time when BG dropped by 5 mg/dl (0.28 mmol/l) from baseline was defined as onset of action. While this procedure allows a precise and reliable determination of onset of action, it requires a lot of experience immediately afterwards when GIR needs to be high enough to bring BG back to the clamp level, but should not be too high to induce an overshoot (i.e. BG levels far above the clamp level). With modern clamp devices it is feasible to establish suitable algorithms for the initial GIR under these conditions. 4.4 Measuring end of action For basal insulins, one of the most important variables is end of action. Treatment regimens, such as once- or twice-daily administration, or potential accumulation in the therapeutic effect in the first treatment days all depend on the duration of action of a basal insulin. The definition for end of action is usually based on the escape in BG levels (without any GIR) towards the end of the clamp in people with diabetes. 21 Initially, a BG threshold of 150 mg/dl (8.3 mmol/l) was used (at a clamp level of 130 mg/dl). 21 This definition was widely used in subsequent clamp studies, but the threshold (and the clamp target) was lowered, 8 so that an increase in the smoothed BG concentrations of as little as 5 mg/dl was defined as end of action. This change was probably attributable to the fact that, with the initial threshold of 150 mg/dl, end of action was often not reached, so that the reported values were right-censored, and end of action was mainly dependent on the duration of the clamp. Even with lower thresholds the duration of action reported in clamp studies tends to be longer than expected from clinical experience. For instance, a recent publication reports mean duration of action (based on the 150 mg/dl threshold) of hours for insulin glargine U100, 22 up to 26 hours for insulin detemir 18 and 13 hours for NPH insulin, 21 which is clearly longer than under clinical conditions. The reason for the rather long duration of action observed in clamp studies is not completely clear, but might be related to an increase in insulin sensitivity during prolonged hyperinsulinaemia or the effect of prolonged fasting Single-dose versus steady-state conditions Basal insulins with ongoing metabolic activity after 24 hours will show some accumulation, that is, a rise in their PK/PD effect over the first treatment days, with a once-daily dosing regimen. It has therefore been proposed that basal insulins should be studied in steady-state rather than single-dose conditions; 3 however, the difference between single-dose and steady-state conditions is quite small for traditional basal insulin analogues such as glargine and detemir, with a duration of action close to 24 hours, so that single-dose data are fairly representative of steady-state conditions. 18,23 By contrast, insulins with a longer half-life and longer duration of action, such as glargine U300 or degludec, will inevitably show some accumulation (i.e. an increase in PK characteristics) over the first treatment days in a once-daily dosing regimen 22,24, although this simple pharmacological principle is not acknowledged by all authors. 25 It should be noted that with these insulins pre-dosing GIR should occur in steady-state conditions because of the ongoing metabolic activity

5 966 HEISE ET AL. A Blood glucose Run-In Period Pre-dose Period 90 mg/dl (5 mmol/l) BG ±20% Insulininfusionrate Study insulin s.c. Glucoseinfusionrate Time (h) B Blood glucose Run-In Period Pre-dose Period 90 mg/dl (5 mmol/l) BG ±20% C Blood glucose 90 mg/dl 5 mmol/l Glucoseinfusionrate Run-in period Insulin infusion rate 2h predosing BG ±20% Study insulin s.c. Onset of Action: Drop in BG by 5 mg/dl Study insulin s.c. Glucoseinfusionrate Time (h) time (h) FIGURE 3 Design of glucose clamp experiments in people with diabetes. After connection of the subjects to the clamp device, their BG levels are adjusted to the clamp target level (here 90 mg/dl or 5 mmol/l). If patients arrive with BG concentrations substantially above this target, an intravenous infusion of human regular insulin is given at a variable rate (A). If the initial BG levels are lower than the target, glucose is infused at a variable rate (B). Patients who receive insulin intravenously will not receive glucose pre-dosing and vice versa. At least 1 hour before the pre-dose period, BG concentrations have to be within a range of 20% of the target. During the pre-dose period, which starts from 2 hours before dosing of study medication, the intravenous infusion of insulin (if any) is lowered as much as possible to keep BG concentrations at the target level without having to infuse glucose (A). Ten minutes before dosing the insulin infusion is tapered off and stopped completely no later than at dosing time. For the determination of onset of action, BG concentrations are allowed to drop by 5 mg/dl from baseline BG levels post-dosing (C). Glucose infusion is started after onset of action occurred and will stabilize BG levels again at the clamp target level. s.c., subcutaneous. of previous insulin injections. Under steady-state conditions, therefore, GIR pre-dosing should not be misinterpreted as carry-over effects of prandial or intravenous insulin. 25 Fortunately, the metabolic activity of previous insulin injections will also avoid or reduce BG elevations prior to the clamp, so that the pre-dosing stabilization of BG at the clamp level is actually easier at steady-state conditions. While, theoretically, individual optimal basal insulin doses (preventing increases in BG without inducing GIR) could be used for steady-state investigations, in practice this will not be feasible because of the high intra-individual PD variability in basal insulins 26,27 and the need to induce some GIR response in glucose clamp studies to obtain meaningful results (see below).

6 HEISE ET AL Insulin dose Glucose clamp studies have investigated a wide range of insulin doses with very low doses of 0.05 U/kg 28 up to at least 1.4 U/kg. 20 While high doses, in particular of fast-acting insulins, pose challenges to the clamper for maintaining good clamp quality, low doses might not elicit any (or only a very small) GIR response, thereby substantially increasing inter-subject (and in case of repeated administrations intrasubject) variability (Figure 4). In general, there is no optimum insulin dose for a PD clamp, but rather the dose should be optimized to achieve the main trial objectives. Low insulin doses are a good choice for the investigation of duration of action. 29 Because of the dose-dependency of the duration of action of insulins (the higher the dose, the longer the duration of action) 20,30 lower doses will help to minimize right-censoring of data. Right-censoring will probably always occur, but the extent is dosedependent: while with a dose of 0.3 U/kg insulin glargine end of action was reached in 65% of T1DM, 29 this number decreased to <15% of clamps in another study with a higher dose of 0.4 U/kg (Profil, Neuss, Germany, unpublished data). Communicating a value for duration of action seems doubtful when this endpoint is only reached in such a small minority of patients. By contrast, if the primary endpoints are based on GIR, higher doses should be used to obtain a robust metabolic response. Typically, doses of at least 0.4 U/kg (for basal insulins) or slightly below 0.1 U/kg (for short-acting insulins) are needed for a robust GIR response. Criticisms that these high doses do not reflect real-life conditions and lead to over-insulinization 3 do not seem to be justified. In our experience, low doses of short-acting insulins, down to 4 U (~0.05 U/kg), can elicit a measurable mean GIR response in T1DM, but with high inter-individual variability. Notably, the results of some glucose clamp studies with low insulin doses could never be reproduced: a completely peakless profile of 0.3 U/kg insulin glargine U could not even be confirmed by the same investigators 23 and GIR (mg/kg/min) Blood Glucose (mg/dl) time (h) FIGURE 4 GIR profile and BG concentrations during a 42-hour clamp in an individual with T1DM after injection of 0.3 U/kg insulin glargine U100. The pharmacodynamic action of the study insulin only elicits a very short and small GIR response, but prevents a pronounced increase in BG concentration during the clamp period. While the chosen dose leads to a near-optimal basal insulin effect under clinical conditions (stabilization of BG concentrations without BG-lowering effect), a higher dose would be needed to induce a meaningful and analysable GIR response an extremely short duration of action seen with 0.35 U/kg insulin detemir (17.5 hours) 31 could not be confirmed in a later study that observed a duration of action of 23.3 hours, despite using a similar clamp methodology and an almost identical insulin dose of 0.4 U/ kg. 18 Thus, low insulin doses with a high variability in the GIR response might be more of a concern under clamp conditions than doses that are higher than those usually used by patients. 4.7 Morning versus evening insulin injection Some authors have demanded that PK/PD study on basal insulins should only be carried out with evening injections 3 based on one of their own glucose clamp studies in T2DM postulating a different PD effect of insulin glargine with morning versus evening injections. 32 However, the credibility of these data remains questionable as interference with endogenous insulin in the T2DM population cannot be ruled out (even though C-peptide differences between study arms were small) and, in particular, as the PK results did not show substantial differences between morning and evening dosings. We analysed the data of two of our own studies in T1DM in which 0.4 U/kg insulin glargine was given at 08:00 hours in one study and at 20:00 hours in the other. GIR profiles are shown in Figure 5 and are remarkably consistent across the two studies. These data strongly argue against an impact of dosing time on the obtained GIR response. Dosing time should therefore be based on logistical considerations and does not have to be taken into account when interpreting PD results. 5 REPRODUCIBILITY OF CLAMP RESULTS The reproducibility of clamp assessments was mainly determined for insulin sensitivity clamps with intravenous insulin infusions. According to Soop et al. 16 the intra-individual coefficient of variation for GIR during these clamps is as low as 5.8%, which is the lowest coefficient of variation ever published for clamp studies. Other studies determined a coefficient of variation ~15%. 33 Some variability will always be observed as there is a biological day-to-day variation in insulin sensitivity that could easily be in the range of 10%-15%. For PD clamps, the observed intra-individual variability will always be a combination of biological changes in insulin sensitivity, the methodological variability and the variability in both insulin absorption and action. The latter can be quite substantial, in particular for basal insulins for which coefficients of variation of up to 99% have been reported for total GIR 34 much higher than the 13%- 18% reported for short-acting insulins. 35 Thus, numerical differences of 10% in PD variables can easily be observed in glucose clamp studies, not only between a biosimilar and the original insulin, 4 but also with repeated administrations of the same insulin. Such a difference therefore should not question the value of the clamp technique and should certainly not be of clinical concern. In general, PD results have been remarkably consistent across glucose clamp studies despite the different techniques (automated versus manual) and the methodological differences outlined earlier. The few clamp studies with irreproducible results (peakless insulin glargine profile, 21 short duration of action for insulin detemir, 19 differences between morning and evening glargine injections 32 and PD

7 968 HEISE ET AL. A GIR (mg/kg/min) Morning Injection Evening Injection Time (h) B GIR (% maximum) Morning Injection Evening Injection Time (h) FIGURE 5 GIR profiles after injection of 0.4 U/kg insulin glargine in people with T1DM. Data were obtained from two different studies: in one study, insulin glargine was injected in the morning at ~08:00 hours (blue curve), in the other study drug injections were performed in the evening at ~20:00 hours. The upper panel (A) shows profiles of GIRs in mg/kg/min (LOESS smoothing, factor 0.25). In the lower panel (B) data were expressed as percent of maximum glucose infusion rates to account for potential differences in insulin sensitivity between the patient populations in the two studies. profiles for glargine and NPH insulin with maximum action of ~20-22 hours in T2DM 31 ) have all been reported by the same group of researchers who often used quite low insulin doses and (as nearly all other clamp investigators) did not provide measures of clamp quality. Otherwise, the reported PD profiles have been very consistent for basal insulins (glargine and detemir 36 and insulin degludec 37,38 ), insulin mixtures 39,40 and short-acting insulins LIMITATIONS OF CLAMP STUDIES While glucose clamp studies can detect even small PD differences between insulins, the clinical relevance of these differences has to be tested in clinical trials. Various attempts to directly calculate the magnitude of BG-lowering effects from GIR results with mathematical models 28 have not led to reliable predictions yet. Nevertheless, the shape of GIR profiles (and the difference in PD effects between various insulins) has been very valuable in predicting clinical outcomes. The faster onset of action of rapid-acting analogues versus human regular insulin shown in numerous clamp studies explains the lower postprandial glycaemic excursions observed clinically. 41 First-generation basal insulin analogues had reduced peak-trough fluctuations, a longer duration of action and lower variability than NPH insulin in glucose clamp studies, which explains the lower hypoglycaemia rate, particularly for nocturnal hypoglycaemia. 42 Likewise, second-generation basal insulin analogues with an even longer duration of action and a lower peak effect further reduce the incidence of (nocturnal) hypoglycaemia. 43 Thus, PD results illustrate the potential of the investigated insulins, but cannot predict the magnitude of potential benefits in clinical studies. This, however, might not be surprising as usually only a small number of highly selected subjects participate in glucose clamp studies. This is another important limitation as these subjects do not necessarily represent the diversity of patients and might easily differ in metabolic control, antidiabetic therapy, insulin sensitivity and other clinical characteristics. This is particularly important for people with T2DM who are quite heterogeneous, especially with regard to individual insulin resistance and endogenous insulin secretion capacity. Thus, clamp results in T2DM cannot be easily related to all people with T2DM, as illustrated by the PD comparison of detemir and glargine in insulin-treated patients with T2DM. 20 Using a clamp duration of 24 hours, that study showed similar PD characteristics, in particular a similar duration of action of the two insulins at doses of 0.4 and 0.8 U/kg (although a trend towards a longer duration of action was seen for glargine with 1.4 U/kg). Clinical studies, however, either showed differences in the proportion of patients with once- versus twice-daily dosing 44 or better diabetes control with glargine in patients on once-daily dosing, 45 indicating that glargine has a longer duration of action than detemir, as demonstrated in clamp studies with T1DM. 36 This discrepancy might be attributable to differences in the patient populations in the clinical studies (insulin-naïve patients with T2DM on one or two oral antidiabetic agents) and the clamp study (insulintreated T2DM with fasting C-peptide 1 nmol/l). In fact, mean fasting C-peptide was nearly twice as high in one of the clinical studies 44 than in the clamp study 20, illustrating how difficult it can be to apply PD results in a small group of T2DM to a large T2DM population. In other populations there are no examples of benefits demonstrated in clinical trials that had been missed in clamp studies. In other words, PD clamps have a very high sensitivity (or might even be oversensitive) in the detection of differences between insulins, so that a lack of PK or PD differences in clamp studies, in particular, if obtained in T1DM, safely exclude clinically relevant disparities. Consequently, the European Medicines Agency guideline for biosimilar insulins permits the waiver of clinical studies if both the physicochemical/functional characterization and the PK/PD profiles indicate similarity between test and reference products. Thus, there are no scientific reasons for challenging the clinical similarity of insulins if bioequivalence has been shown in several clamp trials in both healthy subjects and those with T1DM. 3,4 7 PHARMACOKINETICS Because clamp studies are cumbersome and expensive it is tempting to just focus on PK evaluations 4 ; however, it is the effect rather than insulin concentrations that is relevant for insulin-treated patients, and

8 HEISE ET AL. 969 TABLE 1 Summary of important variables and options in glucose clamp studies Option Advantages Limitations Clamp technique Automated Frequent BG measurements and GIR adjustments ð potential for higher clamp quality Minimized investigator-bias Manual Investigators are able to learn how to improve GIR adjustments during the course of a study Suitable for PD characteristics of all insulins (not dependent on special algorithms) Outdated technology in some devices (Biostator) Algorithms for GIR adjustments have not been validated and might not be optimal for PD characteristics of all insulins Biostator algorithm leads to high oscillations in BG and GIR ð smoothing of GIR curves required BG measurements and GIR adjustments only every 3-10 minutes ð high clamp quality difficult to achieve for fast-acting insulins Potential for investigator bias Study population People with T1DM No interference with endogenous insulin ð particularly important for long-acting insulins People with T2DM Represent largest target population Easier stabilization of pre-dosing BG to target Stimulation of endogenous insulin secretion can usually be avoided in T2DM with low fasting C-peptide (<1 nmol/l) Healthy people Population with highest degree of insulin sensitivity Generally no need for pre-dosing BG stabilization Insulin dose Wash-out of previous insulin therapy and pre-dosing BG stabilization needed Results not easily transferable to people with T2DM Heterogeneous with regard to insulin sensitivity and endogenous insulin secretion capacity ð PD results not easily transferable from a small number of patients to a general population with T2DM Insulin-resistant population ð high insulin doses needed for a robust GIR response Potential interference with endogenous insulin ð not suited for investigations of long-acting insulins with low PD effect No good assessment/correction for endogenous insulin effects possible Low ( 0.1 U/kg for fast-acting, 0.3 U/kg for long-acting insulins) Relevant for daily therapy and particularly for closed-loop systems Assessment of duration of action of long-acting insulins based on BG escape feasible No GIR response in some or even most clamps ð High variability in observed PD characteristics Assessment of very low insulin doses (e.g. <4 U of fast-acting insulins) not reliably feasible in a clamp setting (almost no GIR response) High GIR response in most or all clamps Higher dose than used by most patients under clinical conditions Duration of action longer than under clinical conditions Pre-dosing stabilization of BG to clamp target Intravenous insulin Only option to quickly lower high BG to clamp target Short half-life ð limited or no impact on PK results Careful titration of insulin infusion rate (usually over several hours) necessary to avoid potential carry-over effects on PD results Simultaneous infusion of insulin and glucose should be avoided Glucose infusion Only option to quickly elevate low BG to clamp target Usually only needed for steady-state conditions or in healthy people Stable GIR pre-dose necessary to assess post-dosing PD effects Assessment of onset of action Fall in BG by 5 mg/dl (0.3 mmol/l) Most reliable indicator of start of insulin effect Requires stable pre-dosing BG Abandons principle of glucose clamp (i.e. fixing BG at a target) Difficult stabilization of BG at target after reach of onset of action (risk of GIR overshoot, in particular with very fast-acting insulins) GIR-related parameters (e.g. early t50%girmax) Easy to obtain from conventional PD profiles No standard GIR parameter for onset of action available Do not really reflect onset of action, but rather early metabolic activity Time-related GIR measures often show great variability Smoothing required to reliably assess time-related GIR parameters Comparison of early t50%girmax only feasible if GIRmax is similar

9 970 HEISE ET AL. the time courses of PK and PD profiles are clearly different, with the PK profiles being left-shifted. In addition, there are a number of issues that have to be considered for PK analyses. These are described below. TABLE 1 Continued Option Advantages Limitations Assessment of end of action (for long-acting insulins) No standard BG value to end of action (traditionally 150 mg/dl (8.3 mmol/l), but lower values [e.g.105 mg/dl (5.8 mmol/l)] used recently) Even with low threshold BG values duration of action is longer in clamp studies than under clinical conditions Issue of right-censoring : many patients do not reach end of action in clamp studies, in particular with high insulin doses BG increase at end of clamp Reflects main purpose of long-acting insulins (control of fasting BG) Relatively easy to obtain from conventional PD profiles No standard GIR parameter for end of action available Do not really reflect end of action, but rather late metabolic activity Time-related GIR-parameters often show great variability Smoothing required to reliably assess time-related GIR-parameters Comparison of late t50%girmax only feasible if GIRmax is similar GIR-related parameters (e.g. late t50%girmax) Single-dose versus steady-state conditions Usually requires pre-dosing BG stabilization in people with diabetes Single-dose Easier, no pre-treatment of patients required Reflects PD activity at clinical conditions for insulins with a duration of action of less or close to 24 hours (e.g. fast-acting insulins, glargine U100, detemir) Requires pretreatment of patients with the study insulin which either requires titration or adjustment of short-acting insulins/carbohydrate intake in case of fixed insulin doses Attainment of steady-state should be documented by measuring trough PK levels, if feasible Steady-state Reflects PD activity in clinical conditions for insulins with a duration of action exceeding 24 hours (e.g. degludec, glargine U300) Pre-dosing BG stabilization under clamp conditions usually easier (only GIR needed to compensate the remaining effect of previous injections) 7.1 Cross-reactivity of insulin assays In healthy people or those with T2DM, PK evaluations can easily be affected by endogenous insulin. The attempt to correct measured insulin concentrations by prevailing C-peptide concentrations remains challenging because of the different half-lives of insulin and C-peptide and potential interference of pro-insulin with the C-peptide measurements. 4 While this could be overcome by studies in T1DM, PK comparisons between different insulin molecules remain challenging, as nearly all assays show different cross-reactivities to human insulin and the various analogues. 46 Commercially available specific assays are restricted to human insulin and insulin lispro. Subtraction methods, that is, using two different assays of which one only cross-reacts with, for example, human insulin and the other cross-reacts with both human insulin and the investigated insulin analogue, have led to implausible results in some studies, with pronounced PK differences between equipotent analogues used at identical doses Pharmacokinetic assessment of insulin glargine Pharmacokinetic assessment of insulin glargine has been particularly difficult as glargine forms two metabolically active metabolites M1 (A21-Gly-insulin) and M2 (A21-Gly-des-30B-Thr-insulin). 48 While a specific assay exists, 26,27 it is not commercially available, neither is another analytical method based on immunoaffinity enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS). 49 The latter method also has the disadvantage of a relatively high lower limit of quantification (0.2 ng/ml 49 or 30 pmol/l 8 ), so that measurable concentrations cannot always be obtained in all individuals, 8,29 especially if low doses are used. While some authors have recommended the use of the LC-MS/MS method for insulin glargine PK assessments, 3 the same authors have used an unspecific assay for their own insulin glargine studies, 32 which might highlight the difficulties of obtaining specific PK assessments for this insulin. 7.3 Pharmacokinetic assessments of acylated, albumin-bound insulins Specific assays for acylated, albumin-bound insulins, such as insulin detemir or insulin degludec, are not commercially available, and cross-reactivity of most assays to these insulins (bound or free) is low. In general, assays cannot distinguish between the free (active) and the albumin-bound (metabolically inactive) portions of the insulin, so that the measured concentrations are considerably higher than those of other insulin preparations that do not bind to albumin 27 and do not necessarily correlate with the insulin s metabolic activity. 3

10 HEISE ET AL Insulin antibodies While some, but not all assays are affected by moderate or high levels of insulin antibodies, this issue can usually be overcome with the measurement of free insulin levels with precipitation of immune complexes before analysis unless insulin antibody levels are very high, which has become rare with the use of highly purified insulin preparations for subcutaneous injection. 50 Although PK assessments have some limitations, they certainly do provide valuable information when combined with PD assessments, in particular, if limitations of the clamp technique make it difficult to fully characterize the properties of an insulin (e.g. in case of very long-acting insulin formulations). 8 CONCLUSIONS A number of methodological details (e.g. study population, insulin dose, design of the run-in period, steady-state vs single-dose conditions, and many more) have to be considered for the optimum design of glucose clamp studies. Some of these details and related options are summarized in Table 1. Overall, the exact clamp design depends on the primary objective of a trial and with a well thought-out design, PD characteristics can be determined with great accuracy and precision. While design issues and/or insufficient clamp quality might be the reason for some clamp studies showing irreproducible results, PD results of clamp studies have usually been remarkably consistent and of value to predict clinical outcomes of new insulins, provided, however, that high clamp quality can be achieved. ACKNOWLEDGMENTS The authors are indebted to all individuals that have participated in glucose clamp studies at Profil in Neuss or Mainz, and to the clinic teams at both sites for the immaculate conduct of many thousands of glucose clamp experiments. Special thanks go to Reinhard Becker (Sanofi, Frankfurt, Germany), Hanne Haahr (Novo Nordisk, Søborg, Denmark), and Lutz Heinemann (Profil, Neuss, Germany) for many years of fruitful discussions on the refinement of the glucose clamp technique. Author Contribution All authors substantially contributed to this publication. TH prepared the first draft which was reviewed by all authors. All authors approved the final version of the paper for submission. Funding information No funding was received for this paper. How to cite this article: Heise T, Zijlstra E, Nosek L, Heckermann S, Plum-Mörschel L, Forst T. Euglycaemic glucose clamp: what it can and cannot do, and how to do it, Diabetes Obes Metab 2016, 18, DOI: /dom REFERENCES 1. Becker RHA. Pharmacodynamic evaluation: diabetes methodologies. In: Vogel HG, Maas J, Gebauer A, eds. Drug Discovery and Evaluation: Methods in Clinical Pharmacology. 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