Metabolism of Insulin Glargine After Repeated Daily Subcutaneous Injections in Subjects With Type 2 Diabetes

After approval by an ethics committee and informed written consent, 18 type 2 diabetic subjects underwent a euglycemic glucose clamp study after subcutaneous injection of 0.4 units/kg glargine and 1 week of daily glargine treatment, as previously reported (5). Subjects were fasted throughout the clamp studies. Glargine metabolism was studied in nine subjects (Supplementary Table 1) from whom plasma was still available (5).

The 31-h blood samples (samples after 31 h were not available) were processed for radioimmunoassay determination of plasma concentration of insulin (which detects endogenous insulin plus injected glargine and its metabolites) (5) and for a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay specific for glargine and metabolites 1 (M1) and 2 (M2) (6) (Supplementary Data). The lower limit of quantification for this method was 33 pmol/L for insulin glargine, M1, and M2.

The maximum plasma concentration (C_max), time to reach C_max, and predose plasma concentration were read directly from the plasma concentration-time data for each subject. The area under the concentration-time curve (AUC) between 0 and 31 h (AUC_0–31) was determined using the trapezoidal rule in which concentrations less than the lower limit of quantification were set at zero. Percentage of glargine and M1 was calculated according to the equation: (glargine or M1/total insulin [glargine + M1]) × 100.

Data are expressed as medians (25th and 75th percentiles). Calculations were performed using NCSS 2007 (NCSS, LLC, Kaysville, UT, www.ncss.com).

Plasma glucose was maintained at 101 mg/dL (25th and 75th percentiles, 99 and 103 mg/dL, respectively) as a result of variable glucose infusion (0.47 mg/kg/min [0.26 and 0.82 mg/kg/min]) for 31 h (Supplementary Fig. 1).

Glargine was detected in only five of the nine subjects (Supplementary Fig. 2). In two subjects, the last measurable concentration was at 11 h (25th and 75th percentiles, 53 and 35 pmol/L, respectively), in two other subjects at 13 h (37 pmol/L for both), and in one subject at 18 h (41 pmol/L). M1 was detected in all subjects up to 31 h, whereas M2 was not detected in any subjects at any time point. M1, but not glargine, was detected at baseline (44 pmol/L [19–131 pmol/L]), likely reflecting the metabolism of glargine injected 24 h before. In fact, 24-h postinjection values of M1 were similar to baseline values (60 pmol/L [52–118 pmol/L]). The median glargine pharmacokinetics-AUC_0–31 was 171 pmol · h/L (0 and 314 pmol · h/L), whereas the M1 pharmacokinetics-AUC_0–31 was 2,166 pmol · h/L (1,622 and 4,955 pmol · h/L) (Fig. 1). Glargine C_max was 40 pmol/L (0 and 48 pmol/L), and M1 C_max was 129 pmol/L (75 and 207 pmol/L). Overall, M1 represented 97% (89 and 100%, respectively) of the total amount of insulin detected in blood after injection of glargine, with glargine contributing only 3% (1 and 8%). The pattern of plasma insulin concentration as measured by radioimmunoassay was similar to that of M1 as detected by LC-MS/MS (Supplementary Fig. 1).

In the current study in type 2 diabetic subjects on glargine treatment for 1 week, a subcutaneous injection of a therapeutic dose of insulin glargine resulted in biotransformation to M1, which constituted >90% of circulating insulin, with only a minor presence of the originally injected glargine in plasma (<10%).

To the best of our knowledge, this is the first study reporting the metabolism of insulin glargine in type 2 diabetic subjects using a specific LC-MS/MS assay (6). Overall, the results are in line with recent findings in type 1 diabetes (Bolli et al. [4]). Taken together, the results suggest that in humans, glargine is rapidly metabolized to M1, whereas the originally injected glargine is, if anything, only minimally present in plasma.

These findings in type 2 diabetic subjects are relevant for the interpretation in vivo of the in vitro data reporting greater affinity of glargine for IGF-1R and greater mitogenic potency compared with HI in some malignant cell lines (7). The present studies indicate that in vivo, glargine concentration in plasma is negligible and well below that shown in vitro to demonstrate greater affinity for IGF-1R (3,8) and greater mitogenesis (9). Because glargine is rapidly and nearly totally metabolized to M1, which has effects on mitogenesis no different than HI (3,7), it is unlikely that there is an increased risk of cancer in humans following administration of insulin glargine, at least via IGF-1R stimulation. Therefore, the findings of in vitro studies on mitogenesis using glargine as such (10) are of limited informative value for interpretation of the results of glargine administration in vivo.

We acknowledge that the current study has limitations (i.e., small sample size and a dose of insulin glargine just above the therapeutic range of the subjects studied). Nevertheless, the results are homogeneous in all subjects, consistent with those in type 1 diabetes (Bolli et al. [4]) and with recent findings of no stimulation of IGF-1R bioactivity by plasma of type 2 diabetic subjects who were given glargine doses greater than those described in the current study (11).

In conclusion, after subcutaneous injection of a therapeutic dose in glargine-treated type 2 diabetic subjects, glargine is only transiently, and at minimal concentration, detectable in plasma, whereas its metabolite M1 accounts for most (>90%) of the plasma insulin concentration. Thus, in vivo, glargine does not exert its long-acting metabolic effects directly as glargine but predominantly via its main metabolite M1.

While this article was in proof, an elegant in vitro study was published that showed that glargine displays higher potency than human insulin for stimulation of insulin/IGF-1 hybrid receptors with greater proliferative/antiapoptotic effects in MCF-7 cells. In contrast, M1 and M2 display lower potency than human insulin.

Pierre-Eugene C, Pagesy P, Nguyen TT, Neuillé M, Tschank G, Tennagels N, Hampe C, Issad T. Effect of insulin analogues on insulin/IGF1 hybrid receptors: increased activation by glargine but not by its metabolites M1 and M2. PLoS One 2012;7:e41992.

The measurement in plasma of glargine, M1, and M2 was performed at and supported by sanofi-aventis in Frankfurt, Germany. Editorial support was provided by Tom Claus, PhD, of PPSI (a PAREXEL Company) and funded by sanofi-aventis.

F.P. received honoraria from Eli Lilly & Co. and sanofi-aventis. G.B.B. received honoraria from sanofi-aventis, MannKind, and Eli Lilly & Co. for scientific advising and consulting. A.H. and R.S. are employees of sanofi-aventis Deutschland. No other potential conflicts of interest relevant to this article were reported.

P.L., F.P., P.R., P.Ca., A.M.A., P.Ci., A.H., and R.S. researched the data, contributed to the discussion, and reviewed and edited the manuscript. G.B.B. contributed to the discussion and reviewed and edited the manuscript. C.G.F. researched the data, contributed to the discussion, wrote the manuscript, and reviewed and edited the manuscript. P.L. 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.

Parts of this study were presented at the 71st Scientific Sessions of the American Diabetes Association, San Diego, California, 24–28 June 2011.

This study is dedicated to the subjects with type 2 diabetes who have volunteered for our studies.