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Supplementary Information Titles

Journal: Nature Medicine

Article Title: Characterization of pancreatic NMDA receptors as possible drug targets for diabetes treatment

Corresponding Author: Eckhard Lammert

Supplementary Item & Number

Title or Caption

Supplementary Figure 1

Grin1 silencing in INS1E cells and pancreatic beta cells increases glucose-stimulated insulin secretion (GSIS).

Supplementary Figure 2 Role of NMDARs in islet cell viability, islet insulin content and second phase insulin secretion.

Supplementary Figure 3 Impact of NMDARs on glucose-induced Ca2+ oscillations and requirement of CaMKII activity for the effect of MK-801 on GSIS.

Supplementary Figure 4 Effects of morphinan derivatives on GSIS.

Supplementary Figure 5

Amantadine (AMT) lowers blood glucose concentrations to a lesser extent compared to dextrorphan (DXO).

Supplementary Figure 6 Db/db mice treated with 1 mg ml–1 and 3 mg ml–1 DXM do not differ in body weight, daily food intake and corticosterone concentrations.

Supplementary Figure 7

Consort flow diagram.

Supplementary Figure 8

Proposed mechanism of NMDAR-regulated insulin secretion.

Supplementary Table 1

Baseline demographic and clinical characteristics of the study sample.

Supplementary Table 2 Primary and secondary outcome measurements.

Supplementary Note

Additional information on the clinical study design.

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Supplementary Information Characterization of pancreatic NMDA receptors as possible drug targets for diabetes treatment Jan Marquard1,2,16, Silke Otter1,3,4,16, Alena Welters1-4,16, Alin Stirban5, Annelie Fischer5, Jan Eglinger1,3,4, Diran Herebian2, Olaf Kletke6, Maša Skelin Klemen7, Andraž Stožer7,8, Stephan Wnendt9, Lorenzo Piemonti10, Martin Köhler11, Jorge Ferrer12,13, Bernard Thorens14, Freimut Schliess5, Marjan Slak Rupnik7,8,15, Tim Heise5, Per-Olof Berggren11, Nikolaj Klöcker6, Thomas Meissner2, Ertan Mayatepek2, Daniel Eberhard1, Martin Kragl1,4 & Eckhard Lammert1,3,4

1Institute of Metabolic Physiology, Heinrich Heine University, Düsseldorf, Germany 2Department of General Pediatrics, Neonatology and Pediatric Cardiology, University Children's Hospital Düsseldorf, Düsseldorf, Germany 3Institute for Beta Cell Biology, German Diabetes Center (DDZ), Leibniz Center for Diabetes Research, Düsseldorf, Germany 4German Center for Diabetes Research (DZD e.V.), Helmholtz Zentrum München, Neuherberg, Germany 5Profil Institute for Metabolic Research, Neuss, Germany 6Institute of Neuro- and Sensory Physiology, University Hospital Düsseldorf, Düsseldorf, Germany 7Institute of Physiology, Faculty of Medicine, University of Maribor, Maribor, Slovenia 8Center for Open Innovations and Research Core@UM, University of Maribor, Maribor, Slovenia 9MLM Medical Labs GmbH, Mönchengladbach, Germany 10Diabetes Research Institute (DRI), IRCCS San Raffaele Scientific Institute, Milano, Italy 11The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Stockholm, Sweden 12Department of Medicine, Imperial College London, London, UK 13Genomic Programming of Beta-Cells Laboratory, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas, Barcelona, Spain 14Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland 15Institute of Physiology, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria

16These authors contributed equally to this work.

Correspondence should be addressed to: E.L. (lammert@hhu.de).

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Supplementary Figure 1 Grin1 silencing in INS1E cells and pancreatic beta cells increases glucose-stimulated insulin secretion (GSIS). (a) Representative western blot showing the knockout efficiency of GluN1 in islets from Pdx1-Cre × Grin1 loxP/loxP mice (GluN1 KO islets) compared to islets from Pdx1-Cre × Grin1 loxP/wt mice (control islets); one replicate (data not shown). (b) Western blot showing the knockdown efficiency of GluN1 in INS1E cells transfected with control siRNA and 3 different GluN1 siRNAs; one replicate for siRNA1 (data not shown). (c) Knockdown efficiency of Grin1 in INS1E cells; n = 3 cell samples each. (d,e) Insulin secretion from INS1E cells transfected with siRNAs and GluN1 siRNA2 in combination with GluN1 cDNA (rescue); n = 4–6 cell samples each. (f) Insulin secretion from INS1E cells incubated without and with 10 µM MK-801; n = 4 cell samples each. (g) Representative western blot showing the knockout efficiency of GluN1 in islets from Ins1-Cre × Grin1 loxP/loxP mice (GluN1(beta) KO islets) compared to islets from Ins1-Cre × Grin1 loxP/wt mice (control islets); one replicate (data not shown). (h) Representative X-gal staining of the pancreas (upper image) and brain (lower image) of Ins1-Cre × R26R LacZ mice; Scale bars, 1 mm; two replicates (data not shown). (i) Insulin secretion with islets from male control and GluN1(beta) KO mice; n = 4 islet batches each. Significance tested by one-way ANOVA followed by Dunnett’s multiple comparison test (c,d) or Tukey’s multiple comparison test (e), and Student’s t-test (f,i). *P < 0.05. All values are means ± SD.

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Supplementary Figure 2 Role of NMDARs in islet cell viability, islet insulin content and second phase insulin secretion. (a) Representative LSM images of mouse islets. Scale bars, 50 µm. (b) Area of dead cells of mouse islets treated as described in (a); n = 3 islet batches with 15–16 islets per batch for each group. (c) Insulin content and (d) insulin secretion from mouse islets; n = 4 islet batches each. Same islet batches used for (c) and (d). (e) Insulin secretion from mouse islets incubated with NMDA; n = 4 islet batches each. (f) Insulin secretion from mouse islets incubated with 500 µM tPDC or 500 µM tPDC in combination with 1,000 µM NMDA; n = 4 islet batches each. (g) Dynamic insulin secretion measurement in mouse islets in the absence or presence of 10 µM MK-801; the large peak representing the first phase and the following flat peak representing the second phase of insulin release. Secreted insulin normalized to KCl-stimulated insulin secretion; n = 4 individual measurements each. (h) Insulin secretion from mouse islets; n = 4 islet batches each. (i) Insulin secretion from control islets and islets derived from SK4 KO mice; n = 5 islet batches each. Significance tested by Student’s t-test (b–d,f–i), and one-way ANOVA followed by Dunnett’s multiple comparison test (e). *P < 0.05. All values are means ± SD.

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Supplementary Figure 3 Impact of NMDARs on glucose-induced Ca2+ oscillations and requirement of CaMKII activity for the effect of MK-801 on GSIS. (a) Representative intracellular Ca2+ concentrations in mouse islets; four repetitions (data not shown). (b) Representative intracellular Ca2+ oscillations in a mouse islet; six repetitions from different islet preparations (data not shown). (c) Representative intracellular Ca2+ oscillations in an islet from a GluN1 KO mouse; 30 repetitions from different islet preparations (data not shown). (d,e) Insulin secretion from mouse islets; n = 4 islet batches each. Significance tested by one-way ANOVA followed by Tukey’s multiple comparison test (d,e). *P < 0.05. All values are means ± SD.

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Supplementary Figure 4 Effects of morphinan derivatives on GSIS. (a) Molecular core structure of dextrorotary morphinans. (b) Molecular structure of DXM and its metabolites. (c) Structure of LVP. (d) Insulin secretion from mouse islets incubated with DXM, DXO, HM or MM; n = 3 islet batches each. (e) Insulin secretion from mouse islets incubated with LVP or DXO; n = 4 islet batches each. (f) Insulin secretion from human islets incubated without or with 10 µM DXO. Islet donor: female, 47 years of age, BMI = 20.6 kg m–2, HbA1C = 4.7%; n = 4 islet batches each. Significance tested by one-way ANOVA followed by Dunnett’s multiple comparison test (d) or Tukey’s multiple comparison test (e), and Student’s t-test (f). *P < 0.05. All values are means ± SD.

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Supplementary Figure 5 Amantadine (AMT) lowers blood glucose concentrations to a lesser extent compared to DXO. (a,b) Insulin secretion from mouse islets incubated without or with two different concentrations of AMT; n = 4-5 islet batches each. (c,d) Blood glucose concentrations of mice during IP glucose tolerance tests without and with AMT (50 µg g–1 body weight and 75 µg g–1 body weight); n = 8 male mice each. Doses of AMT ≥ 100 µg g–1 body weight were toxic to the mice. (e) Blood glucose concentrations of mice during an IP glucose tolerance test without and with DXO (50 µg g–1 body weight). Same cohort as in (c); n = 7–8 male mice each. Significance tested by Student’s t-test (a,b), and Student’s t-test with Holm-Bonferroni correction (c–e). *P < 0.05. All values are means ± SD.

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Supplementary Figure 6 Db/db mice treated with 1 mg ml–1 and 3 mg ml–1 DXM do not differ in body weight, daily food intake and corticosterone concentrations. (a) Fasting blood glucose concentrations of male db/db mice treated without or with DXM in drinking water. Stepwise increase of DXM concentrations during the first 3 weeks of treatment from 1 mg ml–1 to 3 mg ml–1 as indicated; n = 8 db/db mice each. (b) Body weight of db/db mice shown in (a); n = 8 db/db mice each. (c) Body weight and (d) daily food intake of male db/db mice continuously treated with 1 mg ml–1 DXM or with 3 mg ml–1 DXM in the drinking water; n = 7 male db/db mice each. (e) Fasting blood glucose concentrations and (f) plasma corticosterone concentrations of db/db mice treated continuously with 1 mg ml–1 DXM or with 3 mg ml–1 DXM in drinking water for three weeks; n = 10 male db/db mice each. Same cohort for (e) and (f). (g) Area of dead cells of human islets incubated without or with a mixture of cytokines (TNF-α, IFN-γ and IL-1β) either alone or in combination with 10 µM DXO; Islet donor: female, 47 years of age, BMI = 29.9 kg m–2; non-diabetic; n = 4 islet batches with 7–10 islets per batch for each group. Significance tested by Student’s t-test with Holm-Bonferroni correction (a–d), Student’s t-test (e,f), and one-way ANOVA followed by Dunnett's multiple comparison test (g). *P < 0.05. All values are means ± SD.

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Supplementary Figure 7 Consort flow diagram.

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Supplementary Figure 8 Proposed mechanism of NMDAR-regulated insulin secretion. Insulin secretory process: Metabolism of glucose increases intracellular ATP and intracellular glutamate concentrations (1). The increased ATP concentrations inhibit ATP-sensitive potassium channels (KATP channels) (2) that normally counteract plasma membrane depolarization by permitting K+ efflux (3). The glucose-dependent, ATP-induced closure of KATP channels triggers plasma membrane depolarization (4), and voltage-dependent calcium channels (VDCCs) become activated (5). As a result, intracellular Ca2+ concentrations increase (6), which induce insulin secretion in a CaMKII-dependent manner (7). Intracellular glutamate concentrations increase in response to glucose metabolism and amplify the insulin secretory process. Counterregulatory process: When glutamate is released from pancreatic beta cells via excitatory amino acid transporters (EAATs) or insulin secretion (8), it also participates in a counter-regulatory process involving NMDA receptors (negative feedback). In addition, pancreatic alpha cells secrete glutamate together with glucagon (9). The extracellular glutamate and glucose-induced plasma membrane depolarization together activate NMDA receptors (10). The latter activate KATP channels (11) and Ca2+-dependent K+ channels (SK4 channels) (12) to counteract plasma membrane depolarization and thus inhibit glucose-stimulated insulin secretion (GSIS) (11,13). By inhibiting the NMDA receptors, MK-801, DXO and its pro-drug DXM (14) prevent this counterregulatory process, prolong the depolarized phases, open VDCCs, and enhance GSIS. Under diabetogenic conditions, NMDAR antagonists also reduce islet cell death by an unknown molecular mechanism.

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Supplementary Table 1 Baseline demographic and clinical characteristics of the study sample. Data are means ± SD (minimum and maximum values), presented in absolute numbers (n) or specified in percent (%).

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Supplementary Table 2 Primary and secondary outcome measurements. Data presented as means ± SD, measured values, absolute numbers (n) or percent (%). Pharmacokinetic (PK) analyses performed with all randomized individuals. Safety analyses included all individuals receiving at least one dose of the study medication. Statistical analysis of the AUC 1–3 h blood glucose (*) as the primary parameter (also shown in Fig. 6g) after log-transformation using a linear mixed model with sequence and treatment as fixed factors and individuals within sequence as a random factor. Comparison of each dose of study medication with placebo. Similar statistical analyses of secondary parameters and other PK parameters. Analyses of safety parameters by means of descriptive statistics.

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Supplementary Note Additional information on the clinical study design. A phase IIa, double-blinded, placebo-controlled, randomized, fourfold crossover study to investigate the glucose lowering effects of DXM and AMT in individuals with T2DM after an oral glucose tolerance test (OGTT) Summary: In this study we investigated the blood glucose (BG) lowering effects of DXM and AMT in 20 male individuals with T2DM on metformin monotherapy; age 59 (46–66) years [mean (range)]; BMI 29.2 (25.2–34.1) kg m–2; HbA1c 6.9 (6.5–7.4)%. Individuals received single oral doses of 60 mg DXM (DXM60), 270 mg DXM (DXM270), 100 mg AMT (AMT100) or placebo and an OGTT one hour (h) after drug intake on four treatment days separated by a washout-period of 7–14 days. DXM270 significantly reduced glycemic excursions (BG-AUC1–5 h) compared to placebo (778.4 vs. 842.9 mg dl–1 h–1, P < 0.01). Both DXM270 and DXM60 increased maximal insulin (INS) concentrations (82.5 and 82.9 vs. placebo 66.4 mU l–1, P < 0.05 respectively) and DXM60 also increased AUCINS(1–5 h) (204.2 vs. placebo 174.0 mU l–1 h–1, P < 0.05). AMT had no significant effect on BG or INS parameters. All adverse events were of mild or moderate intensity, and DXM60 was particularly well tolerated. In sum, DXM, in contrast to AMT, significantly stimulated GSIS and, at a higher dose, also reduced postprandial glycemic excursions. Additional studies are needed to clarify the effects of long-term DXM treatment in individuals with T2DM. Background: Loss of insulin secretory capacity by pancreatic beta cells contributes to the pathogenesis and progression of T2DM. There is currently no therapy available that protects individuals with diabetes from loss of beta cell insulin secretory capacity. This study was performed to determine whether NMDA receptor antagonism, which increases survival of islets in diabetic db/db mice as well as survival of human islets exposed to inflammatory cytokines, exerts anti-hyperglycemic effects in individuals with diabetes following an OGTT and whether it increases serum insulin concentrations. In addition, it was investigated whether a low dose of DXM (60 mg), shown to be well tolerated in humans, also affected serum insulin concentration of individuals with T2DM. If this proves to be the case, NMDA receptors appear to be useful drug targets and some of their antagonists could already be explored in long-term clinical trials. Complete in- and exclusion criteria: Inclusion criteria: 1. Signed and dated written informed consent obtained before any study-related

activities. 2. Male individuals with a diagnosis of T2DM according to American Diabetes

Association (ADA) criteria at least 4 months prior to screening. 3. Medical history without major pathology (with the exception of T2DM). 4. Stable regimen of metformin monotherapy for at least 3 months. 5. Aged between 45 and 70 years of age, both inclusive. 6. Body mass index (BMI) between 25 and 35 kg m–2, both inclusive. 7. HbA1c ≥ 6.5% and < 7.5%. 8. A male individual who is sexually active and not surgically sterilized must agree to

use adequate contraceptive methods as described in Section 4.3.2.1 from the time of first study drug administration until 90 days after last dosing.

9. Ability and willingness to abstain from grapefruit juice (and all grapefruit containing products) throughout the study starting 24 h prior to first study drug administration as well as from alcohol, methylxanthine-containing beverages or food (coffee, tea, Coke, chocolate, “power drinks”), tobacco products and from engaging in strenuous physical activity from 24 h prior to each admission until discharge from the unit.

Exclusion criteria: 1. Individuals with type 1 diabetes, maturity onset diabetes of the young (MODY) or

secondary forms of diabetes such as due to pancreatitis.

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2. Current or previous treatment with insulin therapy (except for treatment within a clinical trial, for surgical procedures or during an acute illness for ≤ 7 days and more than 14 days before the first administration of study drug).

3. Treatment with any hypoglycemic medication other than metformin within the 3 months prior to screening.

4. Individuals with any severe medical or surgical history of conditions likely to confound study assessments or study endpoints, for example but not limited to hemoglobinopathies, inflammatory bowel disease, cystic fibrosis, bariatric surgery or any surgery shortening the intestine, history of galactose intolerance, lactose- or glucose-galactose-malabsorption.

5. Serious respiratory disease, serious or unstable coronary heart disease (unstable angina, myocardial infarction within the preceding 6 months), congestive heart failure of New York Heart Association Class II (NYHA II) or worse (slight limitation of physical activity; comfortable at rest, but ordinary physical activity resulting in fatigue, palpitation, or dyspnea), second and third degree heart block, superior vena cava syndrome, uncontrolled hypertension, history of congenital QT-syndrome within family, history of stroke (within the preceding 6 months) or serious peripheral vascular disease.

6. History of arrhythmia (multifocal premature ventricular contractions, bigeminy, trigeminy, ventricular tachycardia or uncontrolled atrial fibrillation) that is symptomatic or requires treatment (grade 3), left bundle branch block or asymptomatic sustained ventricular tachycardia are not allowed.

7. Marked diabetic complications: severe autonomic or sensory neuropathy including gastroparesis; proliferative retinopathy.

8. Any respiratory disease leading to respiratory insufficiency or respiratory depression including but not limited to: asthma bronchiale, chronic obstructive pulmonary disease.

9. Clinically significant abnormalities of vital signs including known bradycardia with pulse rate < 55 min–1 or 12-lead ECG findings including pre-treatment QTc > 420 msec (if the ECG shows a QTc value of > 420 ms, two further ECGs will be repeated within the next 30 minutes, at least 2 minutes apart, with the mean value of these three consecutive ECGs being conclusive).

10. History of or current prostate hyperplasia. 11. History of or current narrow angle glaucoma. 12. Clinically significant abnormal hematology, biochemistry, lipids, or urinalysis or

coagulation screening tests, as judged by the investigator. 13. Moderate or severe renal dysfunction defined as a calculated glomerular filtration rate

(GFR) < 70 ml min–1 using the Cockcroft-Gault calculation. 14. Clinical or laboratory evidence of hepatic dysfunction or disease; laboratory evidence

defined as any of the following parameters: alkaline phosphatase > twofold upper limit of normal (ULN), alanine transaminase (ALT) > twofold ULN, aspartate transaminase (AST) > twofold ULN or bilirubin > threefold ULN. Isolated mild rise in bilirubin considered to be due to Gilbert’s condition is allowed.

15. Uncontrolled high blood pressure (diastolic blood pressure > 95 mmHg or systolic blood pressure > 160 mmHg), unless clearly documented to be white-coat hypertension.

16. History of any psychiatric condition that might impair the individual’s ability to understand or to comply with the requirements of the study or to provide informed consent.

17. History of relevant drug or food allergies or a history of severe anaphylactic reaction. 18. Smoking more than five cigarettes or cigars or pipes daily and not willing to abstain

from any consume of tobacco products 24 h prior to each admission until discharge. 19. Currently active or history of alcohol abuse (defined as an intake of more than 24

units of alcohol per week; one unit of alcohol equals approximately 250 ml of beer, 100 ml of wine or 35 ml of spirits) or drug addiction (including soft drugs like cannabis products).

20. Positive alcohol test at screening. 21. Use of concomitant medication, which would confound study conduct:

§ Monoamine oxidase (MAO) inhibitors or selective serotonin reuptake inhibitors (SSRI) (Fluoxetine, Paroxetine), any other antipsychotic and

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antidepressant medication or drugs with depressant effects on the central nervous system.

§ Antiarrhythmic therapy class IA (e.g. Chinidine, Disopyramide, Procainamide) and class III (e.g. Sotalol).

§ Antihistaminic therapy (e.g. Astemizole, Terfenadine). § Use of macrolide antibiotics (e.g. Erythromycine, Clarythromycine) and

gyrase inhibitors (e.g. Sparfloxacine). § Use of antimycotic therapy (e.g. Bupidine, Halofantrine, Cotrimoxazole,

Pentamidine, Cisapride, Bepridile). § Use of weight-loss agents. § Medications which have the potential to inhibit Cytochrome P450 (CYP 450)

2D6: Amiodarone, Chinidine, Haloperidole, Paroxetine, Propafenone, Thioridazine, Cimetidine and Ritonavir.

§ Use of any other medication that may influence glycemic control or food intake or body weight as judged by the investigator (except for metformin).

§ Concomitant intake of both Triamterene and Hydrochlorothiazide. § Intake of any medication including over-the-counter medication, herbal and

dietary supplements such as St John’s Wort, vitamins and minerals that could affect the outcome of the study (including glycemic control, food intake or weight), within 2 weeks before the first administration of the study medication or less than five times the half-life of that medication, whichever is longer. Metformin and commonly prescribed medications that are unlikely to affect the outcome of the study are allowed.

22. Use of drugs that have a known risk of causing Torsades de Pointes are prohibited within 14 days prior to randomization (‘Torsades de Pointes List’ on www.azcert.org/medicalpros/drug-lists/bycategory.cfm APPENDIX 2: Drugs that Have a Risk of Causing QT Prolongation).

23. Participation in another clinical trial within the 3 months preceding screening or 5-halflives of the drug studied, whichever is longer, prior to study medication administration.

24. Participation in more than three other clinical trials in the 10 months preceding screening.

25. Malignancy within 5 years of study start, except for successfully treated local basal cell carcinomas.

26. Individuals known to be positive for Hepatitis B surface antigen or Hepatitis C antibodies (or diagnosed with active hepatitis according to local practice).

27. Positive result to the screening test for Human Immunodeficiency Virus (HIV)-1 antibodies, HIV-2 antibodies or HIV-1 antigen according to locally used diagnostic tests.

28. Use of concomitant medication which would likely interact with metformin (according to the subject information leaflet).

29. Individuals who have donated or lost more than 500 ml blood within 3 months prior to screening.

30. History of hypersensitivity to the study medication or any of the excipients or to medicinal products with similar chemical structures.

31. Veins unsuitable for repeated venipuncture. Complete secondary parameters: Secondary pharmacodynamic parameters: AUCBG(0–1h) area under the blood glucose concentration-time profile from 0–1 h post-dose

(i.e. before starting the OGTT) AUCBG(1–5 h) area under the blood glucose concentration-time profile from 1–5 h post-dose

(i.e. 0–4 h after starting the OGTT) AUCBG(1–1.5 h) area under the blood glucose concentration-time profile from 1–1.5 h post-

dose (i.e. from 0–30 min after starting the OGTT) ΔBGmax maximum blood glucose excursion after starting the OGTT BGmax maximum blood glucose concentration after starting the OGTT tBGmax time to maximum blood glucose concentration after starting the OGTT AUCINS(0–1 h) area under the serum insulin concentration-time profile from 0–1 h post-dose (i.e. before starting the OGTT)

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AUCINS(1–1.5 h) area under the serum insulin concentration-time profile from 1–1.5 h post-dose (i.e. from 0–30 min after starting the OGTT)

AUCINS(1–5 h) area under the serum insulin concentration-time profile from 1–5 h post-dose (i.e. from 0–4 h after starting the OGTT)

AUCINS(1–3 h) area under the serum insulin concentration-time profile from 1–3 h post-dose (i.e. from 0–2 h after starting the OGTT)

INSmax maximum serum insulin concentration after starting the OGTT tINSmax time to maximum serum insulin concentration after starting the OGTT The analysis for glucagon concentration was performed post-hoc: AUCGLUCAGON(0–1 h) area under the blood glucagon concentration-time profile from 0–1 h

post-dose (i.e. before starting the OGTT) AUCGLUCAGON(1–1.5 h) area under the plasma glucagon concentration-time profile from 1–

1.5 h post-dose (i.e. 0–30 min after starting the OGTT) AUCGLUCAGON(1–3 h) area under the plasma glucagon concentration-time profile from 1–3

h post-dose (i.e. from 0–2 h after starting the OGTT) AUCGLUCAGON(1–5 h) area under the plasma glucagon concentration-time profile from 1–5

h post-dose (i.e. from 0–4 h after starting the OGTT) GLUCAGONmax maximum plasma glucagon concentration after starting the OGTT Secondary pharmacokinetic parameters: AUCDXM (0–1 h) area under the plasma DXM concentration-time profile from 0–1 h post-dose

(i.e. before starting the OGTT) AUCDXM (1–5 h) area under the plasma DXM concentration-time profile from 1–5 h post-dose

(i.e. from 0–4 h after starting the OGTT) AUCHM (0–1 h) area under the plasma HM concentration-time profile from 0–1 h post-dose

(i.e. before starting the OGTT) AUCHM (1–5 h) area under the plasma HM concentration-time profile from 1–5 h post-dose

(i.e. from 0–4 h after starting the OGTT) AUCDXO (0–1 h) area under the plasma DXO concentration-time profile from 0–1 h post-dose

(i.e. before starting the OGTT) AUCDXO (1–5 h) area under the plasma DXO concentration-time profile from 1–5 h post-dose

(i.e. from 0–4 h after starting the OGTT) AUCMM (0–1 h) area under the plasma MM concentration-time profile from 0–1 h post-dose

(i.e. before starting the OGTT) AUCMM (1–5 h) area under the plasma MM concentration-time profile from 1–5 h post-dose

(i.e. from 0–4 h after starting the OGTT) Primary and secondary outcome measurements in Supplementary Table 2. tBGmax, tINSmax, AUCHM (0–1 h), AUCHM (1–5 h), AUCMM (0–1 h), AUCMM (1–5 h) can be obtained upon request. Randomization: Each individual received each of the four possible treatments according to the randomization scheme: Dosing Visit 1 Dosing Visit 2 Dosing Visit 3 Dosing Visit 4 Sequence 1 DXM 60 mg AMT 100 mg DXM 270 mg Placebo Sequence 2 AMT 100 mg Placebo DXM 60 mg DXM 270 mg Sequence 3 DXM 270 mg DXM 60 mg Placebo AMT 100 mg Sequence 4 Placebo DXM 270 mg AMT 100 mg DXM 60 mg

Randomization numbers used in this study were: 101–120. Replacement numbers used in this study were: 201–220. Re-replacement numbers used in this study were: 301–320 Eligible individuals were assigned the lowest available randomization number upon receipt and investigator’s evaluation of the safety laboratory results. The Qualified Person was

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informed of that randomization number in order to prepare and dispense the appropriate treatment. Each individual received one treatment during each dosing visit. There was a maximum of four treatments for each individual. Individuals would have to be replaced to ensure that at least 20 individuals completed this study.

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