Study Design

This was a single-site (Profil, Neuss, Germany), phase 1, exploratory, open-label, two-period, fixed-sequence, multiple-dose study (Supplemental Fig. 1). The study consisted of four study periods: a screening and run-in period, a glargine U100 treatment period, an efsitora treatment period, and a safety follow-up.

During the glargine and efsitora treatment periods, there were three hypoglycemia provocation periods: a 24-h prolonged fast, a 24-h prolonged fast and exercise, and following a double dose. The trial is registered on clinicaltrials.gov (NCT04957914).

The trial was conducted in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice guidelines and was approved by the Ethics Committee of the North Rhine Medical Association (protocol reference number: 202051). All participants provided written informed consent prior to participation.

Study Population

Eligible participants included adults (18–70 years) with T2D on a stable regimen of daily basal insulin (NPH insulin, glargine U100 or U300, insulin detemir, or degludec) with or without metformin, dipeptidyl peptidase IV inhibitors, sodium-glucose cotransporter 2 inhibitors, or glucagon-like peptide 1 agonists for at least 3 months. Participants had a BMI between 18.5 and 40 kg/m2 and HbA1c levels ≥ 6.5 and ≤ 9.5% and had not experienced any episodes of severe hypoglycemia or hypoglycemia unawareness in the 6 months prior to screening.

Study ProceduresScreening and Run-In

During the run-in period, self-monitored plasma glucose (SMPG) assessments were conducted on 4 of the 7 days prior to the first treatment period visit. On day −1, participants were admitted to the clinical research unit (CRU) and received a continuous glucose monitoring (CGM) system (Dexcom G6) to aid in the safe conduct of the study; CGM was not used for any safety endpoints. Participants were unblinded to the CGM, with preset alarms used for ‘urgent low’ (plasma glucose (PG) ≤ 55 mg/dL), ‘urgent low soon’ (PG ≤ 55 mg/dL within 20 min), ‘low’ (PG ≤ 70 mg/dL), and ‘fall rate’ (PG dropping at −3 mg/dL per minute) alerts.

Treatment Period 1—Glargine

Participants transitioned from their pre-study basal insulin to glargine U100 and received the first dose of glargine in the evening of day 1. Participants were discharged from the CRU the following day. Glargine was self-administered daily while outside the CRU. Dose assessments, including safety, CGM, and SMPG reviews, were conducted weekly. Glargine was titrated based on the median fasting plasma glucose (FPG) from SMPG assessed during the prior week to achieve a target fasting glucose of ≤ 100 mg/dL (Supplemental Table 1). On day 22, participants were required to have a stable FPG between 80 and 150 mg/dL based on three median FPG readings from the prior week in order to continue in the study.

The three hypoglycemia provocation tests occurred while in the CRU. The 24-h prolonged fast began on day 27 after the participants received a dose of glargine and ate an evening meal. Participants were discharged 2 days later. The prolonged fast followed by exercise provocation began on day 33. Again, participants received a dose of glargine and ate a meal prior to the start of a 24-h prolonged fast. The following morning, participants exercised for 40 min (on a stationary bicycle, with a target heart rate of 40% of heart rate reserve (HRR)). On days 35 through 37, participants transitioned from evening to morning dosing of glargine. Specifically, participants received 50% of the daily insulin glargine dose in the evening of day 35, in the morning on day 36, and in the evening on day 36. Finally, participants received a full daily glargine dose in the morning of day 37. The double-dosing provocation began with an overnight fast in the evening of day 37. The following morning, participants received a double dose of glargine and then ate a predefined meal. The daily dose of glargine was continued the next day. Participants were discharged from the CRU 2 days later.

On days 42 and 45, participants had outpatient visits to complete safety assessments and to review the CGM and SMPG data.

Treatment Period 2—Efsitora

On day 50, participants re-entered the CRU to begin the transition from glargine to efsitora. For the first efsitora injection, a one-time starting dose, calculated based on the glargine daily dose and the median baseline FPG (Supplemental Table 2), was administered on day 51. Participants were discharged the following day. Efsitora was administered weekly at the CRU by site personnel. Outpatient dose assessments, including safety, CGM, and SMPG reviews, were conducted weekly from day 58 through 100. Efsitora dose titrations were performed based on the median FPG from the previous week to achieve a fasting glucose target range of 80–130 mg/dL (Supplemental Table 3).

Similar to the glargine treatment period, the hypoglycemia provocation tests occurred while the participants were admitted to the CRU. The 24-h prolonged fast began on day 105 after participants ate an evening meal. The participants were discharged 2 days later. The prolonged fast with exercise provocation began on day 111 after the participants ate an evening meal. The following morning, the participants exercised for 40 min. On day 113, for the double-dosing provocation test, the participants ate an evening meal and began an overnight fast. Participants then received a double dose of efsitora the following morning. Participants were discharged on day 118.

On day 121, participants reviewed their CGM and SMPG data, completed safety assessments, and began their transition back to pre-study basal insulin based on the SMPG, hypoglycemia data, and investigator discretion.

Follow-up

At least 75 days after the last dose of efsitora, participants returned to the CRU for a follow-up visit.

Hypoglycemia treatment

Since one main goal of the study was to understand potential differences in hypoglycemia in response to the provocations, the usual management and intervention practices for hypoglycemia were not followed. Instead, during the hypoglycemia provocation periods for both efsitora and glargine, participants were monitored for signs and symptoms of hypoglycemia throughout the fasting period. PG was monitored approximately every 6 h, avoiding postprandial measurements, using the Super GL glucose analyzer. PG was monitored more frequently after the first PG reading below 100 mg/dL, a CGM hypoglycemia alarm was triggered, or the participant reported symptoms of hypoglycemia. PG was monitored every 15 min following a glucose reading below 80 mg/dL and monitored every 10 to 15 min if PG fell below 70 mg/dL. PG was monitored more frequently if the PG dropped by 3 mg/dL per min or more and triggered the CGM alarm. It was only when the participant experienced intolerable symptoms or PG was < 54 mg/dL that 15 g of oral carbohydrates was administered, and the participant was monitored for 90 min.

Study Assessments and Outcome Measures

The PD parameter assessments included nadir PG, time to hypoglycemia, the duration of hypoglycemia events occurring during the provocation monitoring periods, and FPG from SMPG. The hypoglycemia incidence and event rates during provocation periods were calculated. The hypoglycemic events were categorized according to the American Diabetes Association (ADA) descriptions as either level 1 hypoglycemia (glucose < 70 mg/dL (3.9 mmol/L) and ≥ 54 mg/dL (3.0 mmol/L)), level 2 hypoglycemia (glucose < 54 mg/dL (3.0 mmol/L)), and level 3 hypoglycemia (severe hypoglycemia characterized by altered mental and/or physical status requiring assistance). Nocturnal hypoglycemia was defined as a hypoglycemia event that occurred at night and presumably during sleep. All hypoglycemic events with a confirmed glucose reading of < 70 mg/dL were recorded. The end time of a hypoglycemic event was recorded when the PG value returned to 70 mg/dL and remained at or above 70 mg/dL for at least 15 min.

Because treatment of hypoglycemia could have included glucose administration, which disrupts the fasting status during the fasting and fasting with exercise provocation tests, the summary of the nadir PG and duration of hypoglycemia for each of these two provocation periods included data for the hypoglycemic events that occurred prior to the first hypoglycemic event that required glucose treatment. For the double-dosing period, during which participants received regular meals, a summary of all events, regardless of the time relative to glucose treatment, was included.

Two sensitivity analyses were conducted. The first assessed the incidence of hypoglycemia in a subgroup of participants who achieved a stable FPG of 80 to 120 mg/dL prior to the provocation periods. The second sensitivity analysis was conducted on a subgroup of participants who had similar FPG values (within 15% of the mean of the two FPG values) in their glargine and efsitora treatments prior to the double-dosing provocation period.

Adverse events (AEs), serious AEs (SAEs), and product complaints (PCs) were reported by participants throughout the study. Additionally, laboratory tests, vital signs, and electrocardiograms were documented.

Statistical AnalysisSample Size

The sample size was determined such that we needed ~ 50 participants to enroll and ~ 40 participants would complete the study. Forty completers have at least 90% power to detect a treatment difference in the incidence of hypoglycemia (< 70 mg/dL) during the double-dose provocation period, assuming an incidence of hypoglycemia of 43% for efsitora, an incidence of hypoglycemia of 91% for glargine, a two-sided significance level of 0.05, and a within-patient correlation of zero between two treatment visits.

The primary objective—the incidence of hypoglycemia during each provocation period—was reported for each treatment, the difference between treatments was calculated, and the 95% confidence intervals were determined using the Wald method with continuity correction. Hypoglycemic provocation monitoring periods were defined as follows:

1.

The prolonged fasting period started when the fast began and ended 24 h later.

2.

The prolonged fasting with exercise period started when the fast began and ended 24 h later.

3.

The double-dosing period started when the double dose was administered and ended 96 h later for efsitora and 48 h later for glargine.

AE safety data were summarized using descriptive statistics.