Original article Different insulin concentrations in resuspended vs. unsuspended NPH insulin: Practical aspects of subcutaneous injection in patients with diabetes P. Lucidi, F. Porcellati, A. Marinelli Andreoli, P. Candeloro, P. Cioli, G.B. Bolli *, C.G. Fanelli Section of Internal Medicine, Endocrinology and Metabolism, Department of Medicine, Perugia University Medical School, Perugia, Italy Introduction The invention of insulin protamine was first reported by Hans Christian Hagedorn in 1936 [1]. Neutral protamine Hagedorn (NPH) insulin was then introduced into the market in 1946 for the treatment of patients with diabetes mellitus. Since then, NPH has continued to be a popularly used insulin worldwide. It is the oldest basal insulin on the market, and is used either on its own, or admixed (with syringes) or premixed (pen cartridges) with soluble insulin. NPH is still used by patients with type 1 or type 2 diabetes despite the availability of modern alternatives, such as long-acting insulin analogues [2]. NPH is an insoluble insulin obtained by co-crystallization of insulin with zinc in the presence of the basic polyarginine peptide protamine at an isophane ratio and neutral pH. The exact binding mode of the insulin–protamine complex is not known [3]. Divalent ions like zinc, and other additive ligands like phenolic derivatives and protamine, along with the type and morphology of the precipitate, all play a role in the slow release of insulin from the precipitated NPH insulin crystals after subcutaneous (s.c.) injection [4]. While the exact mechanisms of the separation of soluble insulin from NPH insulin crystals are also not known, all manufacturers suggest that tipping the vial or pen cartridge 10– 20 times is sufficient for homogeneous resuspension. To improve the efficiency of the process, they have placed miniaturized beads in the cartridges to aid the mixing [5]. However, Jehle et al. [6] have demonstrated that up to 91% of NPH-treated patients either do not perform the resuspension procedure or do it inappropriately, with deleterious consequences for blood glucose control. NPH is characterized by highly variable pharmacokinetics (PK) and pharmacodynamics (PD) in patients with diabetes [2,7]. Such variability could be due, in theory, to a variable mechanism of the precipitation of crystals in s.c. tissue after injection and/or the Diabetes & Metabolism xxx (2017) xxx–xxx A R T I C L E I N F O Article history: Received 11 May 2017 Accepted 15 May 2017 Available online xxx Keywords: NPH insulin NPH resuspension Premixed insulin A B S T R A C T Aims. – This study measured the insulin concentration (Ins [C] ) of NPH insulin in vials and cartridges from different companies after either resuspension (R+) or not (R; in the clear/cloudy phases of unsuspended NPH). Methods. – Measurements included Ins [C] in NPH(R+) and in the clear/cloudy phases of NPH(R), and the time needed to resuspend NPH and time for NPH(R+) to separate again into clear/cloudy parts. Results. – In vials of NPH(R+) (assumed to be 100%), Ins [C] in the clear phase of NPH(R) was < 1%, but 230  41% and 234  54% in the cloudy phases of Novo Nordisk and Eli Lilly NPH, respectively. Likewise, in pen cartridges, Ins [C] in the clear phase of NPH(R) was < 1%, but 182  33%, 204  22% and 229  62% in the cloudy phases of Novo, Lilly and Sanofi NPH. Time needed to resuspend NPH (spent in tipping) in vials was brief with both Novo (5  1 s) and Lilly NPH (6  1 s), but longer with all pen cartridges (50  8 s, 40  6 s and 30  4 s from Novo, Lilly and Sanofi, respectively; P = 0.022). Time required for 50% separation into cloudy and clear parts of NPH was longer with Novo (60  7 min) vs. Lilly (18  3 min) in vials (P = 0.021), and affected by temperature, but not by the different diameter sizes of the vials. With pen cartridges, separation into clear and cloudy parts was significantly faster than in vials (P < 0.01). Conclusion. – Ins [C] in NPH preparations varies depending on their resuspension or not. Thus, subcutaneous injection of the same number of units of NPH in patients with diabetes may deliver different amounts of insulin depending on its prior NPH resuspension.  C 2017 Elsevier Masson SAS. All rights reserved. * Corresponding author. Department of Medicine, Perugia University Medical School, Hospital Santa Maria della Misericordia, Torre Ellittica, floor +1, room #17, 06129 Perugia, Italy. Fax: +39 075 578 344 4. E-mail address: geremia.bolli@unipg.it (G.B. Bolli). G Model DIABET-892; No. of Pages 5 Please cite this article in press as: Lucidi P, et al. Different insulin concentrations in resuspended vs. unsuspended NPH insulin: Practical aspects of subcutaneous injection in patients with diabetes. Diabetes Metab (2017), http://dx.doi.org/10.1016/j.diabet.2017.05.004 Available online at ScienceDirect www.sciencedirect.com http://dx.doi.org/10.1016/j.diabet.2017.05.004 1262-3636/  C 2017 Elsevier Masson SAS. All rights reserved.