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Diabetes Care Volume 41, March 2018 531

Ulrike Hovelmann,¨ 1 Britta Væver Bysted,2 Pharmacokinetic and Ulrik Mouritzen,2 Francesca Macchi,2 Daniela Lamers,1 Birgit Kronshage,1 Pharmacodynamic Characteristics Daniel´ Vega Møller,2 and Tim Heise1 of , a Novel Soluble and Stable Analog Care 2018;41:531–537 | https://doi.org/10.2337/dc17-1402

OBJECTIVE Treatment of severe outside of the hospital setting is limited to glu- cagon formulations requiring reconstitution before use, which may lead to erroneous

or delayed glucagon administration. We compared the pharmacokinetic (PK) and THERAPEUTICS AND TECHNOLOGIES EMERGING pharmacodynamic (PD) characteristics and safety and tolerability of different doses of dasiglucagon, a novel soluble glucagon analog, with approved pediatric and full doses of GlucaGen in -induced hypoglycemia in patients with .

RESEARCH DESIGN AND METHODS In this single-center, randomized, double-blind trial, 58 patients with type 1 diabetes received single subcutaneous injections of 0.1, 0.3, 0.6, or 1.0 mg dasiglucagon or 0.5 or 1.0 mg GlucaGen in a state of hypoglycemia ( target 55 mg/dL) induced by an intravenous insulin infusion.

RESULTS Dasiglucagon demonstrated a dose-dependent and rapid increase in plasma concen- trations, reaching a maximum at ∼35 min with a half-life of ∼0.5 h. Dasiglucagon rapidly increased plasma glucose (PG) by ‡20 mg/dL (9–14 min) to PG ‡70 mg/dL (within 6–10 min), similar to GlucaGen, but with a longer-lasting and greater effect on PG. All patients on both treatments reached these end points within 30 min (pre- 1Profil, Neuss, Germany defined success criteria). Both treatments were well tolerated. was the most 2Zealand Pharma A/S, Glostrup, frequent adverse event, occurring at a similar rate (44–56%). Corresponding author: Ulrike Hovelmann,¨ ulrike .hoevelmann@profil.com. CONCLUSIONS Received 31 July 2017 and accepted 22 Novem- Dasiglucagon was well tolerated and showed an early PD response similar to that of ber 2017. GlucaGen at corresponding doses, suggesting comparable clinical effects of the two reg. no. NCT02660008, clinicaltrials glucagon formulations. Dasiglucagon has the potential to become an effective and .gov. reliable rescue treatment for severe hypoglycemia in a ready-to-use pen. This article contains Supplementary Data online at http://care.diabetesjournals.org/lookup/ suppl/doi:10.2337/dc17-1402/-/DC1. Currently available glucagon formulations for rescue treatment of severe hypoglycemia This article is featured in a podcast available at require reconstitution of dry powder in aqueous solution immediately prior to each use. http://www.diabetesjournals.org/content/ The process of reconstitution and delivery is complex and requires adequate education of diabetes-core-update-podcasts. families and caregivers, which is not ideal for an emergency drug. Despite training, the © 2017 by the American Diabetes Association. reconstitution process could still lead to erroneous or delayed administration of glucagon, Readers may use this article as long as the work is properly cited, the use is educational and not at least when used by medical nonprofessionals in stressful emergency situations (1,2). for profit, and the work is not altered. More infor- Native glucagon is a highly unstable prone to spontaneous polymerization mation is available at http://www.diabetesjournals and formation of amyloid-like fibrils, resulting in the product becoming unusable within .org/content/license. 532 PK/PD of Novel Stable Liquid Glucagon Analog Diabetes Care Volume 41, March 2018

1 day of reconstitution (3). Instructions with those of GlucaGen different dose screening visit, a screening visit (3–30 days for commercially available glucagon allow ranges in insulin-induced hypoglycemia in before the first dosing visit), one dosing only immediate usage after reconstitution patients with type 1 diabetes. visit for group 1 and two dosing visits for (4,5). Stable liquid formulations of a glu- groups 2–4 separated by 7 6 3days cagon analog in a ready-to-use injection RESEARCH DESIGN AND METHODS washout, and a follow-up visit (21 6 3 device would offer major clinical advan- Trial Design days after the last dosing visit). In group tages, such as speed and ease of use for This was a single-center (Profil, Neuss, 1, patients received a single s.c. dose of rescue treatment in patients experienc- Germany), randomized, double-blind trial 0.1 mg dasiglucagon (1 mg/mL, liquid for- ing severe hypoglycemia. Furthermore, a in patients with type 1 diabetes. The trial mulation in prefilled ; Zealand simplified glucagon application might re- included four groups of patients, with the Pharma, Copenhagen, Denmark) or a sin- duce the fear of hypoglycemic events, first eight patients (group 1) randomly gle s.c. dose of lyophilized glucagon (1 mg which is sometimes the underlying source allocated (3:1) to either a mini-dose of for reconstitution, GlucaGen; Novo Nor- of suboptimal glycemic control in patients dasiglucagon (0.1 mg) (6 patients) or a full disk, Copenhagen, Denmark). In groups with diabetes, resulting in an increased dose (1.0 mg) of GlucaGen (2 patients). 2–4, patients were administered three risk for complications (6). Subsequent patients were randomly allo- different single s.c. doses for dasigluca- Bihormonal artificial systems cated to one of the three other treatment gon and two different single doses for might be another promising option for a groups (groups 2–4, with 16 patients in GlucaGen. Both treatments were received stable glucagon analog. Maintaining each group) and received a single dose in a randomized sequence. euglycemia in artificial pancreas settings of 0.3 mg (group 2), 0.6 mg (group 3), or For maintenance of double blinding, is challenging because of the slow onset 1.0 mg (group 4) dasiglucagon and, in a the appropriate dose/volume was trans- and the relative long duration of action cross-over fashion, a single pediatric dose ferred from the prefilled syringes (dasi- of subcutaneous (s.c.) prandial , of GlucaGen of 0.5 mg (group 2) or full dose glucagon) or from the vial filled with so a more aggressive insulin titration could of 1.0 mg (groups 3 and 4) (Supplementary freshly reconstituted solution (GlucaGen) easily lead to hypoglycemia. Hypoglycemia Fig. 2). (Pediatric dose of GlucaGen has into 1-mL disposable syringes with at- could be avoided with an s.c. glucagon for- been approved for children ,25 kg or tached 27 G needles (Becton Dickinson) mulation in the bihormonal artificial pan- younger than 6–8 years of age.) by staff not otherwise involved in trial pro- creas systems, but currently available The trial protocol was reviewed and cedures. Both trial products were adminis- glucagon formulations are only stable for approved by the local health authority tered by s.c. injection into a lifted skinfold 24 h. Undoubtedly, a glucagon analog with (Bundesinstitut fur¨ Arzneimittel und Medizin- of the abdominal wall around the umbili- longer stability at body temperature would produkte) and by an independent ethics cus. Basal insulin was continued as usual substantially increase the feasibility of bi- committee (Arztekammer¨ Nordrhein). The during the dosing day, whereas short-acting hormonal pump delivery devices (7–14). trial was performed in accordance with the insulin was replaced by Dasiglucagon is a novel stable peptide Declaration of Helsinki and the Interna- (Apidra; Sanofi Deutschland GmbH, Frankfurt, analog of human glucagon in an aqueous tional Conference on Harmonization and Germany) from 12 h prior to each dosing solution at neutral pH, consisting of 29 Good Clinical Practice. Written informed onwards. Patients using continuous s.c. in- amino acids with 7 amino acid substitutions consent was obtained before initiation of sulin infusion continued their basal insulin relative to native glucagon. (Dasiglucagon any trial-related activities. The trial was reg- rate during the experiment. is the proposed international nonpro- istered at ClinicalTrials.gov (trial identifier: Patients attended the clinical site in the prietary name.) These amino acid substi- NCT02660008). morning after an overnight fast and par- tutions result in improved physical and ticipated in a manual hypoglycemic clamp Participants chemical stability compared with cur- Eligible adults were aged between 18 and procedure that started with a variable in- rently available glucagon formulations 50 years, both inclusive, had been diag- fusion of intravenous (i.v.) insulin glulisine (Supplementary Fig. 1). nosed with type 1 diabetes per American (15 units Apidra dissolved in 49 mL saline ’ Dasiglucagon can be dissolved to at Diabetes Association criteria, and had been and 1 mL of the patient s own blood to least 20 mg/mL at pH 7.0 in the presence treated with insulin for $12 months (15). prevent insulin to tubing mate- and absence of preservatives (m-cresol). Participants were required to have a gly- rial), targeting a blood glucose level of 6 Ongoing stability studies show stability at cosylated (HbA ) ,8.5% 55 mg/dL 10%, corresponding to a 1c 6 40°C under shaking conditions with absence (69.4 mmol/mol) and body weight be- plasma glucose (PG) level of 62 mg/dL fi fl fl of brillation in a Thio avin T uorescence tween 60 and 90 kg (both inclusive). Pa- 10% (3.4 mmol/L) prior to dose adminis- m , assay (samples containing 40 mol/L tients were excluded if they had clinically tration. If PG levels decreased 56 mg/dL fl Thio avin T, excitation 450 nm, and emis- significant concomitant , had clin- (3.1 mmol/L) prior to dosing, i.v. glucose sion 485 nm) for at least 7 days, while na- ically significant abnormal values in clini- was infused and the run-in period ex- fi tive glucagon in the same assay brillated cal laboratory screening tests, were tended until the target range was estab- within 3 h, enabling the use of dasiglucagon habitual smokers, or had any other con- lished for at least 10 min before dose in a ready-to-use rescue device and poten- dition conflicting with trial participation administration. After dose administra- tial use in pump delivery devices. or evaluation of study results. tion, subjects with hypoglycemic PG The objectives of the current study were concentrations ,56 mg/dL were immedi- to compare the pharmacokinetic (PK) and Procedures ately treated with i.v. glucose until a PG pharmacodynamic (PD) properties as well The trial consisted of an informed con- value of .70 mg/dL (3.9 mmol/L) was as safety and tolerability of dasiglucagon sent visit obtained at least 1 day prior to established. care.diabetesjournals.org Hovelmann¨ and Associates 533

The PK and PD effects of the study drugs [AUE]0–30min) were analyzed as well as PG sampling intervals, it showed neither normal were assessed over 360 min postdosing excursions 30 min postdose (CE30min). For nor log-normal distribution and was there- with plasma samples for the determina- correction for endogenous glucagon con- fore analyzed using the Wilcoxon signed tion of dasiglucagon/glucagon and glucose centrations, glucagon concentrations were rank test for paired observations within being taken predose and then every 5 min baseline adjusted (BL). Other PK measures each group. In addition, point estimates from dosing until 40 min postdose fol- comprised the total (AUC0–360min and for median differences between treat- lowed by at 50, 60, 75, 100, 150, 200, AUC0–inf)(AUC0–inf is defined as area under ments and corresponding 90% CIs were 260, 300, and 360 min postdose. the plasma dasiglucagon concentration determined using Hodges and Lehmann vs. time curve from 0 to infinity, whereas procedure. Assessments it is calculated from baseline-adjusted and As lz and related parameters (t1/2, Determination of dasiglucagon in human truncated for GlucaGen profiles with a cut- total body clearance, volume of distri- plasma was done by use of a validated an- off at 2.5 h) and maximum (Cmax)plasma bution, and mean residence time) showed alytical method using off-line and on-line dasiglucagon and glucagon[BL] concentra- markedly skewed distributions with gluca- solid phase extraction and liquid chroma- tions, time to maximum (tmax), terminal gon[BL] concentrations, these end points tography with tandem mass spectrometric elimination rate constant (lz), terminal were calculated from baseline-adjusted detection, which had a lower limit of quan- plasma elimination half-life (t1/2), total glucagon profiles truncated at 2.5 h in a tification (LLOQ) of 10.0 pmol/L. Glucagon body clearance, volume of distribution, post hoc analysis. No statistical compari- was determined using a validated radioim- and mean residence time. sons were done with these parameters. munoassay (Euro Diagnostica AB, Malmo,¨ PDeffectswereanalyzedwithuseofto- Dose proportionality of Cmax and AUCs Sweden) performed on a 1470 Wizard Au- tal PG area under effect curves (AUE0–last), of dasiglucagon were analyzed using a re- tomatic Gamma Counter (PerkinElmer) with maximum PG concentration effect above gression analysis with the log-transformed an LLOQ of 4.7 pmol/L. baseline (CE), and tmax excursion. Key sec- end points as response and log dose as PG was determined with a validated ondary endpoints evaluated successcriteria fixed effect. If 1 was included in the colorimetric assay (hexokinase/glucose- for glucose rescue such as the time to 95% CIs of the estimated slope of the re- 6-phosphate dehydrogenase test kit for achieve a PG $70 mg/dL (3.9 mmol/L) gression line, dose proportionality was glucose; Roche Diagnostics, Mannheim, and the time to an increase of $20 mg/dL assumed. Germany), performed on a MODULAR (1.1 mmol/L), as well as the proportion of EVO/P-Module (Roche Diagnostics) with patients reaching these goals within 30 min RESULTS an LLOQ of 2.9 mg/dL (0.16 mmol/L). Dur- after dosing. Subject Disposition and Characteristics ing insulin-induced hypoglycemia, blood A total of 76 patients were screened and glucose levels were monitored closely Statistical Analyses 58 subjects were randomized and treated on-site using a laboratory glucose analyzer No formal sample size calculation was with trial products. Two patients with- (Super GL glucose analyzer; Dr. Muller¨ performed for this study, which was drew consent after the first dosing visit Geratebau¨ GmbH, Freital, Germany). A aimed at providing a first insight on the (from group 2, GlucaGen 0.5 mg, and validated ELISA method was used for PK/PD properties of dasiglucagon in a group 3, dasiglucagon 0.6 mg). Fifty-six the detection of IgG- and IgM-ZP4207/ new, optimized formulation. patients completed the trial. One patient glucagon antibodies in human serum All statistical analyses were performed (group 1) was excluded from both PK and (YBS, York, U.K.). The sensitivity of the using SAS System for Windows, version 9.4 PD analyses owing to a postdose hypogly- assays was 13.6 and 11.8 ng/mL for the (SAS Institute, Cary, NC). PK parameters cemic event treated with i.v. glucose in- anti-ZP4207 and anti-glucagon methods, were calculated with WinNonlin, version fusion. Therefore, 57 exposed patients respectively. 6.4 (Pharsight Corporation, Mountain were included in the PK and PD analysis Safety assessments included adverse View, CA). (full analysis set) and 58 exposed patients fi events, hypoglycemic episodes (de ned The primary PK and PD end points were included in the safety analysis set. , as PG 56 mg/dL), local tolerability at (AUC0–30min,AUC0–360min,Cmax,andtmax One PK and PD data set from one visit the injection site (assessed predose and and AUE0–30min,AUE,CE30min,CE,and (group 4) was excluded from analysis, as 30, 120, and 360 min postdose), labora- tmax) for group 1 were analyzed descrip- PD measurements were missing between tory safety parameters, vital signs and tively. For groups 3 and 4, data for 1.0 mg 10 and 50 min. All groups were compara- electrocardiogram (assessed predose GlucaGen were pooled across groups for ble with regard to age, weight, height, and 30 and 360 min postdose), physical summary statistics. PK/PD end points and BMI (Supplementary Table 1). examination, and anti-drug antibody were log transformed and compared be- measurements (samples taken prior to tween treatments with a linear model PK Results start of each insulin-induced hypoglyce- ANOVA with treatment, period, sequence, The PK profile of dasiglucagon was char- mic procedure and at follow-up). and patient within sequence as fixed ef- acterized by a dose-dependent and rapid fects. Least squares (LS)-mean values of increase in plasma levels. Dasiglucagon End Points dasiglucagon and GlucaGen as well as reached maximum plasma concentrations For evaluation of early PK and PD effects, the differences of the means and 90% later than GlucaGen (35 vs. 20 min, re- partial areas under the curve (AUCs) CIs were estimated and backtransformed spectively, based on medians over all da- of plasma dasiglucagon and glucagon (exponentially transformed) in order to siglucagon and all GlucaGen doses). The concentrations (AUC0–30min) and PG con- find the estimated ratios and CIs of re- half-life of dasiglucagon was ;0.5 h centrations (area under the effect curve sponses. As tmax was dependent on (Table 1 and Fig. 1). Total exposure 534 PK/PD of Novel Stable Liquid Glucagon Analog Diabetes Care Volume 41, March 2018

Table 1—PK data Dasiglucagon dose GlucaGen dose 0.1 mg 0.3 mg 0.6 mg 1.0 mg 0.5 mg 1.0 mg N 5 1616161733

AUC0–30min (pmol * h/L) 99.4 (32.0) 302 (78.9) 444 (163) 884 (307) 375 (104) 600 (180)

AUC0–360min (pmol * h/L) 451 (123) 1,360 (166) 2,630 (368) 4,800 (697)

AUC0–360min,BL (pmol * h/L) 939 (177) 1,660 (315)

AUC0–inf (pmol * h/L) 451 (123) 1,360 (166) 2,640 (365) 4,810 (696)

AUC0–inf,BL,Trunc (pmol * h/L) 895 (169) 1,630 (311)

Cmax (pmol/L) 334 (113) 976 (208) 1,570 (445) 2,800 (767)

Cmax,BL (pmol/L) 1,100 (307) 1,720 (526)

tmax (h) 0.50 (0.5–0.6) 0.63 (0.3–0.8) 0.58 (0.5–1.7) 0.63 (0.3–0.8) 0.25 (0.2–0.6) 0.37 (0.2–0.8)

t1/2 (h) 0.43 (0.09) 0.43 (0.07) 0.49 (0.14) 0.54 (0.17)

t1/2,BL,Trunc (h) 0.37 (0.07) 0.42 (0.13)

Data are mean 6 SD unless otherwise indicated; tmax shows median (range). GlucaGen AUC0–30min,AUC0–360min,andCmax are shown calculated from baseline-adjusted data, whereas AUC0–inf and t1/2 are displayed as calculated from baseline-adjusted and truncated (Trunc) GlucaGen profiles with a cutoff at 2.5 h.

(AUC0–inf) at compared dose levels was PD Results and Fig. 2), there were no differences in higher for dasiglucagon compared with Despite later PK tmax, the early PD re- the median time to achieve PG $70 GlucaGen, while Cmax values were compa- sponse (AUE0–30min and CE30min)of mg/dL: 6 min for dasiglucagon doses of rable. Treatment ratios for AUC0–inf,BL for 0.3 mg dasiglucagon was comparable $0.3 mg and 6–7 min for both GlucaGen 0.3 mg and 0.6 mg dasiglucagon versus with that of 0.5 mg GlucaGen, as were doses (10 min for 0.1 mg dasiglucagon). 0.5 and 1.0 mg GlucaGen administration the early PD responses of 0.6 mg dasiglu- All patients achieved PG $70 mg/dL were 1.46 (90% CI 1.213; 1.752) and 1.59 cagon and 1.0 mg GlucaGen. Treatment within 30 min after dosing across all treat- (1.299; 1.950), respectively, whereas ratios for dasiglucagon versus GlucaGen ments and doses. Median time to reach a treatment ratios for Cmax,BL for 0.3 and are shown for PD end points in Table 3. PG increase of 20 mg/dL was 9–10 min for 0.6 mg dasiglucagon versus 0.5 and The overall effect in terms of AUE was dasiglucagon doses of $0.3 mg, similar to 1.0 mg GlucaGen administration were higher with 0.3 mg dasiglucagon versus the 10 min observed with both GlucaGen 0.91 (90% CI 0.817; 1.007) and 1.03 0.5 mg GlucaGen (P , 0.0001) and for doses (14 min for 0.1 mg dasiglucagon) (0.885; 1.199), respectively. Dasiglucagon both 0.6 mg (P = 0.0043) and 1.0 mg (Table 2). met the dose-proportionality criteria for (P , 0.0001) dasiglucagon versus 1.0 mg Safety AUC0–30,AUC0–360, and AUC0–inf,sodose GlucaGen. Time to maximum PG in- proportionality can be assumed even creased with increasing dose levels, as All doses of dasiglucagon were safe and well tolerated. Gastrointestinal side ef- though for Cmax the upper limit of the observed with both treatments. While fects occurred with a similar frequency 95% CI was slightly ,1 (Supplementary median tmax values trended to be higher Table 2). for dasiglucagon versus GlucaGen (Table 2 after dasiglucagon and GlucaGen treat- ments, and the most frequent treatment- emergent adverse event (TEAE) was nausea, accounting for 53 of the total of 143 TEAEs observed. A considerable pro- portion of these patients also experi- enced vomiting (22) (in most cases 2–3h postdose). The second most frequent TEAE was , accounting for 30 of 143 TEAEs in 27 patients. While there were numerically more with dasiglucagon (40 vs. 15%), there were no indications of dose dependency of adverse events for either dasiglucagon or GlucaGen (Supplementary Table 3). Headache occurred most often (50%) in the dasiglucagon 0.1 mg group, whereas the incidence in the 1-mg dose groups was relatively small (dasiglucagon 31% and GlucaGen 21%). Postdose hypoglyce- Figure 1—PK profiles. Mean plasma concentration profiles and SEM after single s.c. doses of mic events occurred infrequently with ei- dasiglucagon and GlucaGen. ther treatment (5 events in 4 patients care.diabetesjournals.org Hovelmann¨ and Associates 535

Table 2—PD data Dasiglucagon dose GlucaGen dose 0.1 mg (N = 5) 0.3 mg (N = 16) 0.6 mg (N =17) 1.0 mg (N =16) 0.5 mg (N = 17) 1.0 mg (N =33)

AUE0–30min (mg * h/dL) 12.9 (5.21) 20.9 (6.13) 21.1 (6.10) 24.1 (5.18) 22.1 (5.48) 21.9 (5.74) AUE (mg * h/dL) 344 (149) 666 (247) 788 (165) 895 (213) 462 (273) 566 (232)

CE30min (mg/dL) 66.1 (23.8) 93.4 (23.7) 98.2 (25.0) 100 (20.3) 93.5 (21.4) 96.5 (21.9) CE (mg/dL) 334 (113) 174 (44.6) 190 (32.2) 209 (40.2) 142 (42.6) 166 (42.5)

tmax (h) 1.25 (0.8–1.7) 1.67 (1.0–2.5) 1.67 (1.7–4.3) 2.50 (1.7–2.5) 1.0 (0.7–5.0) 1.25 (0.8–6.1) Dasiglucagon dose GlucaGen 0.1 mg (N = 5) 0.3 mg (N =16) 0.6mg(N = 17) 1.0 mg (N = 16) 0.5 mg (N =15) 1.0mg(N = 31) Time to reach PG levels $70 mg/dL (min) 10.0 (2.0–17.0) 6.0 (0–13.0) 6.0 (0–9.0) 6.0 (0–9.0) 6.0 (0–9.0) 7.0 (0–10.0) Time to increase in PG levels $20 mg/dL (min) 14.0 (11.0– 10.0 (7.0–20.0) 9.0 (6.0–16.0) 9.0 (7.0–15.0) 10.0 (6.0–13.0) 10.0 (5.0–15.0) 27.0)

Data are mean 6 SD or median (minimum–maximum), except for tmax, which shows median (range). with dasiglucagon and 9 events in 8 pa- PG increase of 20 mg/dL and time to clinical relevance, in particular in settings tients with GlucaGen). Four events oc- reach PG $70 mg/dL were similar for that are more typical for daily clinical life curred within 2 h postdosing (2 events dasiglucagon and GlucaGen across the than the i.v. induction of hypoglycemia each with 0.1 mg dasiglucagon and 1.0 mg tested doses, as were AUE0–30min and with insulin, e.g., after strenuous physical GlucaGen). These events might be mostly CE30min (Table 2). activity or after s.c. injection of a high in- attributed to a protracted blood glucose This indicates that dasiglucagon and sulin dose. decline after the induction of hypogly- GlucaGen have similar early PD properties Stability of dasiglucagon has been cemia with i.v. insulin. Seven additional to treat insulin-induced hypoglycemia. It demonstrated in an extensive evaluation events were observed between 4 and is worth pointing out that the overall gly- program. Publication of these results is 6 h postdosing with GlucaGen, whereas cemic response over the 6-h observation under way. In general, substitution of the other three hypoglycemic events period, as represented by AUE and CE 7 out of 29 amino acids could potentially with dasiglucagon occurred .100 h (Table 2), was higher with dasiglucagon result in changed specificity in biological postdosing. for all three comparisons with GlucaGen, action and in immunogenic reactions. No serious adverse event occurred, mostly due to higher PG concentrations However, anti-drug antibodies related to and all adverse events were either of observed subsequent to the 1-h time dasiglucagon treatment have not been mild (113 events) or moderate (30 events) point after dosing (Table 2). The higher observed in phase 1 or 2 clinical trials, intensity. total and longer-lasting glucose response and there were not any indications of Local tolerability findings were rare (7 with dasiglucagon suggests not only a neutralizing antibodies in the preclinical findings in 5 patients with dasiglucagon higher biopotency of dasiglucagon versus or clinical development program. Further and 5 findings in 4 patients with GlucaGen) GlucaGen but also that the longer-lasting long-term data are needed to further as- and mild, and all disappeared within effect of dasiglucagon could potentially sess immunogenic reactions. The benefits 30 min postdosing. No anti-drug antibod- reduce the risk of recurrent hypoglycemia of a stable, easy-to-use glucagon formu- ies were detected. after administration as rescue medica- lation are undisputed, and other ap- All doses of dasiglucagon were conse- tion. This speculative view may be sup- proaches are in development to achieve quently considered safe and well tolerated. ported by the observation that no recurrent this goal (10–14). One of the first de- hypoglycemic events occurred in the velopments reported by Chabenne and CONCLUSIONS time period of 4–6 h after dasiglucagon colleagues (12,13) uses a pH adjustment In this study, dasiglucagon at all dose lev- in contrast to seven events observed after and backbone stabilization to achieve els consistently and quickly reestablished GlucaGen administration. However, clini- aqueous solubility and stability. Another euglycemia after insulin-induced hypogly- cal investigations are needed to show development is an intranasal glucagon cemia in adults with type 1 diabetes. whether these PD differences between releasing 3 mg glucagon powder into Most importantly, the time to reach a dasiglucagon and GlucaGen are of any the nose after pushing of a small plunger on the bottom of the device. While re- Table 3—Treatment comparisons constitution is not required before use, the formulation contains phospholipid Treatment comparisons N Treatment ratios 90% CI dodecylphosphocholine as absorption AUE0–30min enhancer and b-cyclodextrine as bulking Dasiglucagon 0.3 mg vs. GlucaGen 0.5 mg 16/17 0.934 (0.8042; 1.0858) agent. The nasal administration was re- Dasiglucagon 0.6 mg vs. GlucaGen 1.0 mg 17/16 0.965 (0.8613; 1.0822) ported to have a higher rate of head/ CE30min Dasiglucagon 0.3 mg vs. GlucaGen 0.5 mg 16/17 1.01 (0.8933; 1.1353) facial discomfort (25% vs. 9% with intra- Dasiglucagon 0.6 mg vs. GlucaGen 1.0 mg 17/16 1.01 (0.9255; 1.1020) muscularly administered (i.m.) GlucaGen (16,17). Published data on this intranasal 536 PK/PD of Novel Stable Liquid Glucagon Analog Diabetes Care Volume 41, March 2018

insulin-induced hypoglycemia that would have been more pronounced in healthy subjects who are rarely or never exposed to hypoglycemia (26–28). Hypoglycemia was induced with an i.v. insulin infusion under tightly controlled conditions, which is a well-established and ethical way to induce hypoglycemia in clinical trials. A controlled setting is also needed to es- tablish comparable baseline conditions across treatmentsda prerequisite for valid comparisons in a study like ours with a limited sample size. This is in particular important for prevailing insulin levels, as high insulin concentrations have been shown to partially prevent glucagon from increasing endogenous glucose pro- duction and thereby affecting glucagon efficacy (29). We therefore used a vari- able i.v. insulin infusion titrated to induc- Figure 2—PD profiles. Mean PG concentration profiles and SEM after single s.c. doses of dasiglu- ing (but not running below) the desired cagon and GlucaGen. hypoglycemic PG target concentration, and we succeeded in establishing similar approach are still scarce, but a noninferiority comparison with glucagon used as hypogly- insulin concentrations across treatments trial indicated a slightly (;3–5 min) slower cemia rescue therapy, glucagon doses in the in our study. As advantageous as this de- rise in PG with nasal glucagon compared artificial pancreas setting are much smaller sign was for creating similar baseline con- with i.m. GlucaGen (18). Our study used and have been reported to be effective in ditions, this setup is different from the s.c. GlucaGen and did not include an i.m. the management of mild or expected forth- usual clinical causes of severe hypoglyce- comparator. However, the mean time coming hypoglycemia in a range of 20–150 mia, which most often comprise of insulin to a PG increase of 20 mg/dL in our study mg in children and adolescents with dosing errors, , and alcohol con- (9–10 min with dasiglucagon) compares type 1 diabetes (22,23). Likewise, low- sumption (30). A constraint of the current favorably with the reported data for dose glucagon boluses of 100–300 mg study is that the use of different analytic both nasal glucagon (16 min) and i.m. were demonstrated to effectively in- methods for GlucaGen (radioimmunoassay) GlucaGen (13 min). Nevertheless, across- crease PG levels of 54 mg/dL after insu- and dasiglucagon (liquid chromatography– study comparisons are always difficult, lin overdosing in pump users (24). In line mass spectrometry) might limit a direct and head-to-head comparisons would with these findings, another study dem- comparison of PK values. However, the be needed to assess a potential faster rise onstrated clinically relevant rises in blood observed PK differences were consistent in PG concentration with dasiglucagon ver- glucose levels with glucagon doses of with the observed PD effects, and the use sus intranasal glucagon application. The 0.11–1.00 mg administered in euglycemic of a specific assay for dasiglucagon could main difficulty with currently marketed glu- or hypoglycemic baseline conditions (25). also be regarded as an advantage, as any cagon products is the need for reconstitution These data and the clear need for lower interference with endogenous glucagon and handling issues, in particular for un- glucagon doses in an artificial pancreas could be avoided. trained medical nonprofessionals (2), again setting made us investigate low doses In addition to the controlled, but artifi- highlighting the importance of an easy-to- of dasiglucagon and a pediatric dose of cial, experimental design, the small sam- use glucagon formulation. Indeed, the GlucaGen in this study. Our results con- ple size is a limitation of this study. While rate of successful glucagon rescue injec- firm that even these low doses of dasiglu- larger clinical trials are needed, the cho- tions was substantially higher in both ex- cagon (100 or 300 mg) efficiently raise PG sen sample size in combination with the perienced and training-na¨ıve (to glucagon concentrations from hypoglycemia, mak- crossover design (thereby excluding inter- injections) caregivers in a study per- ing it potentially usable in a bihormonal individual confounders [29]) was suffi- formed with the use of an auto-injector artificial pancreas setting and also a viable cient to demonstrate small differences (G-Pen) compared with using the currently treatment option for mild hypoglycemia. in, for example, time to maximum con- available glucagon kits (19). The auto- Strengths of the current trial comprise centrations and total exposure. The clini- injector solution was preferred by all par- the of patients with type 1 di- cal relevance of these differences needs ticipants in this human factors study. abetes, who are the most sensitive and to be explored in future trials. Furthermore, a stable, liquid glucagon relevant target population for hypoglyce- In conclusion, our study shows that the formulation would also enable the devel- mia rescue therapy. Furthermore, a study novel glucagon analog dasiglucagon in a opment of bihormonal artificial pancreas in people with type 1 diabetes avoids the stable liquid formulation quickly and ef- systems. Safety and efficacy of such sys- confounding influence of endogenous in- fectively reestablished euglycemia after tems have been demonstrated in adults, sulin and minimizes the influence of coun- insulin-induced hypoglycemia at the tested adolescents, and children (20,21). In terregulatory hormonal responses to doses from 0.1 to 1.0 mg in adults with care.diabetesjournals.org Hovelmann¨ and Associates 537

type 1 diabetes. In comparison with 4258/SPC/GlucaGen+Hypokit+1+mg/. Accessed glucagon for treatment of insulin-induced hypoglyce- GlucaGen, dasiglucagon needed similar 9 July 2017 mia in adults with type 1 diabetes: a randomized time to increase PG by 20 mg/dL or to 5. Glucagon for Injection. Lilliy USA [article on- crossover noninferiority study. Diabetes Care 2016; line], Available from http://www.lillyglucagon 39:264–270. Diabetes Care 2016;39:e193–e194 reach PG $70 mg/dL, and the overall .com/assets/pdf/glucagon_brochure.pdf. Ac- 19. Newswanger B, Prestrelki S, Cummins M, rise in PG levels was slightly longer lasting cessed 9 July 2017 Andre A, Ching K. Formative, comparative human and higher. With these characteristics, 6. Wood JR, Miller KM, Maahs DM, et al.; T1D factors study of a glucagon auto-injector vs. recon- dasiglucagon is a promising candidate Exchange Clinic Network. Most youth with stitutedglucagoninasimulatedseverehypoglyce- type 1 diabetes in the T1D Exchange Clinic Reg- mia rescue situation (Abstract) [Internet], 2016. for hypoglycemia rescue therapy. Further istry do not meet American Diabetes Association Available from http://online.liebertpub.com/doi/ clinical trials confirming this potential are or International Society for Pediatric and Adoles- pdf/10.1089/dia.2016.2525. Accessed 9 July 2017 under way. cent Diabetes clinical guidelines. Diabetes Care 20. Haidar A, Legault L, Matteau-Pelletier L, et al. 2013;36:2035–2037 Outpatient overnight glucose control with dual- 7. Jacobs PG, El Youssef J, Castle J, et al. Auto- artificial pancreas, single-hormone ar- Duality of Interest. This study was funded by mated control of an adaptive bihormonal, dual-sensor tificial pancreas, or conventional Zealand Pharma A/S, Denmark. B.V.B., U.M., and artificial pancreas and evaluation during inpatient therapy in children and adolescents with type 1 F.M. are employees of Zealand Pharma A/S, and studies. IEEE Trans Biomed Eng 2014;61:2569–2581 diabetes: an open-label, randomised controlled D.V.M. was an employee of Zealand Pharma A/S 8. El-Khatib FH, Balliro C, Hillard MA, et al. Home trial. Lancet Diabetes Endocrinol 2015;3:595–604 during trial conduct. T.H. is a shareholder use of a bihormonal bionic pancreas versus insulin 21. Haidar A, Legault L, Messier V, Mitre TM, of Profil, which received research funds from pump therapy in adults with type 1 diabetes: Leroux C, Rabasa-Lhoret R. Comparison of dual- Adocia, Biocon, Dance Pharmaceuticals, , a multicentre randomised crossover trial. Lancet hormone artificial pancreas, single-hormone arti- Johnson & Johnson, Julphar, Medimmune, 2017;389:369–380 ficial pancreas, and conventional insulin pump Mylan, Nordic Bioscience, , Poxel, 9. Russell SJ, El-Khatib FH, Sinha M, et al. Outpa- therapy for glycaemic control in patients with Roche Diagnostics, Saniona, Sanofi, Senseonics, Sky- tient glycemic control with a bionic pancreas in type 1 diabetes: an open-label randomised con- ePharma, and Zealand Pharma. In addition, T.H. type 1 diabetes. N Engl J Med 2014;371:313–325 trolled crossover trial. Lancet Diabetes Endocrinol is a member of advisory panels for Novo Nordisk 10. Pohl R, Li M, Krasner A, De Souza E. Develop- 2015;3:17–26 and received speaker honoraria and travel grants ment of stable liquid glucagon formulations for 22. Haymond MW, Schreiner B. Mini-dose gluca- from Eli Lilly, Novo Nordisk, and Sanofi.Noother use in artificial pancreas. J Diabetes Sci Technol gon rescue for hypoglycemia in children with potential conflicts of interest relevant to this article 2015;9:8–16 type 1 diabetes. Diabetes Care 2001;24:643–645 were reported. 11. Steiner SS, Li M, Hauser R, Pohl R. Stabilized 23. Hartley M, Thomsett MJ, Cotterill AM. Mini- Author Contributions. U.H. contributed to glucagon formulation for bihormonal pump use. dose glucagon rescue for mild hypoglycaemia in study design, researched data, wrote the man- J Diabetes Sci Technol 2010;4:1332–1337 children with type 1 diabetes: the Brisbane expe- uscript, and reviewed and edited the manuscript. 12. Chabenne JR, DiMarchi MA, Gelfanov VM, rience. J Paediatr Child Health 2006;42:108–111 B.V.B., U.M., D.V.M., and T.H. contributed to DiMarchi RD. Optimization of the native glucagon 24. Ranjan A, Schmidt S, Madsbad S, Holst JJ, study design, researched data, and reviewed and sequence for medicinal purposes. J Diabetes Sci Nørgaard K. Effects of subcutaneous, low-dose edited the manuscript. F.M., D.L., and B.K. Technol 2010;4:1322–1331 glucagon on insulin-induced mild hypoglycaemia contributed to data research and reviewed and 13. Chabenne J, Chabenne MD, Zhao Y, et al. A in patients with insulin pump treated type 1 di- edited the manuscript. B.K. performed the statis- glucagon analog chemically stabilized for immedi- abetes. Diabetes Obes Metab 2016;18:410–418 tical analysis. All authors reviewed the manuscript ate treatment of life-threatening hypoglycemia. 25. Blauw H, Wendl I, DeVries JH, Heise T, Jax T; and approved it for submission. U.H. and T.H. are Mol Metab 2014;3:293–300 PCDIAB consortium. Pharmacokinetics and phar- the guarantors of this work and, as such, had full 14. Newswanger B, Ammons S, Phadnis N, et al. macodynamics of various glucagon dosages at dif- access to all the data in the study and take respon- Development of a highly stable, nonaqueous glu- ferent blood glucose levels. Diabetes Obes Metab sibility for the integrity of the data and the accuracy cagon formulation for delivery via infusion pump 2016;18:34–39 of the data analysis. systems. J Diabetes Sci Technol 2015;9:24–33 26. Lecavalier L, Bolli G, Cryer P, Gerich J. 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