Multicenter, Open-Label, Two-Arm, Pilot Trial for Safety Reduction of Basal Insulin Dose Combined with SGLT2 Inhibitor in Type 1 Diabetes Mellitus: Study Protocol for RISING-STAR.

Background: SGLT2 inhibitor combined with insulin therapy is a novel therapy for patients with type 1 diabetes mellitus. Without the reduction of basal insulin, hypoglycemia could occur frequently in this therapy. But diabetic ketoacidosis is an undesirable adverse effect in case with basal insulin reduction. The aim of this study is to explore whether the reduction of the basal insulin dose combined with SGLT2 inhibitor in patients with type 1 diabetes mellitus can reduce the frequency of hypoglycemia and be used safely. We hypothesized that with an adequate basal insulin dose, the frequency of hypoglycemia is higher if the basal insulin dose is not reduced when combined with SGLT2 inhibitor. Methods and Analysis: The study has a two-arm design; 60 subjects with type 1 diabetes mellitus are being recruited from 7 hospitals. The basal insulin dose before the start of the SGLT2 inhibitor combination therapy is the reference. Study subjects are stratied into two groups based on the ratio of basal insulin daily dose (Basal) to total daily insulin dose (TDD). The subjects are instructed to reduce the basal insulin dose by 10% or 0% for Basal to TDD ratio of <0.4 and > 0.4, respectively. The primary outcome is the frequency of hypoglycemia per day during the intervention period (administration of SGLT2 inhibitor) as determined by self-monitoring of blood glucose (SMBG). The secondary outcome is the frequency of ketosis before and after the intervention. Discussion: 10% basal insulin reduction could reduce hypoglycemia as well as could not increase ketosis in case that the ratio of basal insulin daily dose to total daily insulin dose is 0.4 or higher, which improve the ecacy and safety of SGLT2 inhibitor treatment patients with type 1 diabetes mellitus. Ethics and Dissemination: The study was approved by Kyoto Prefectural University of Medicine, Clinical Research Review Board (CRB5180001). The results will be disseminated through presentations at appropriate conferences and meetings, and published in peer-reviewed journals.


Background
Type 1 diabetes mellitus (T1DM) is a disorder characterized by absolute insulin de ciency, mainly due to autoimmune-mediated pancreatic β-cell destruction.Although the cause of pancreatic β-cell destruction is not completely elucidated, susceptibility genes and environmental factors have been implicated [1].The number of patients with T1DM with absolute insulin de ciency is estimated at approximately 100,000-140,000 in Japan.The age of onset is mainly in childhood to adolescence, and in Japan, the incidence rate in persons aged 0-19 years is 4.4/1000 [2].
In Japan, oral diabetic medication is not covered by the national health insurance program, making intensive insulin therapy the principal treatment for T1DM.The insulin dose should be self-titrated based on the quantity of carbohydrate ingested.Therefore, therapy with only insulin may cause increased body weight, especially in T1DM patients with excess carbohydrate intake, which increases the risk of macrovascular complications.On the contrary, although low-carbohydrate diets can reduce the total insulin dose and the number of self-injections, they could result in nutritional imbalances.Increased mean body mass index (BMI) reported in T1DM patients [3] additionally increases the risk of cardiovascular disorders.Sodium-glucose co-transporter (SGLT)-2 inhibitors reduce hyperglycemia via insulin-independent mechanisms by increasing glucose elimination via the kidneys [4].Although oral anti-diabetic drugs that do not affect insulin secretion have been thought to be effective in the treatment of T1DM and in the prevention of diabetic complications, they have not previously been approved for T1DM.Recently, an SGLT2 inhibitor, dapagli ozin, in combination with insulin therapy, was approved for T1DM [4].
Dapagli ozin selectively inhibits SGLT2 located in the proximal renal tubule, which promotes the excretion of glucose in urine by suppressing the reabsorption of glucose.This action lowers plasma glucose concentration.Since the plasma glucose lowering effect is insulin-independent, SGLT2 inhibitors cause hypoglycemia less frequently, and lead to body weight loss in T1DM patients [5].In addition, since SGLT2 inhibitors prevent cardiovascular complications in patients with type 2 diabetes mellitus [6], these bene ts are also expected in T1DM patients [7].
Hypoglycemia is one of the important determinants of glycemic control in the treatment of T1DM [8].Many patients with T1DM struggle to achieve glycemic control and experience signi cant uctuations in blood glucose levels despite insulin treatment [4,9].Across randomized trials, SGLT2 inhibitors demonstrated signi cant reductions in glycated hemoglobin, glucose exposure, and measures of glycemic variability, as well as increased time in the target glycemic range when administered as an adjunct to insulin [4].
To prevent hypoglycemia, many T1DM patients might reduce the basal insulin dose, which might impact sugar control.While the addition of SGLT2 inhibitors might reduce the risk of hypoglycemia, their use has been reported to increase the frequency of diabetic ketoacidosis [10].Therefore, the reduction of the insulin dose to prevent hypoglycemia during adjunctive SGLT2 inhibitor treatment may increase the risk of ketoacidosis, whereas maintaining the same insulin dose in order to prevent ketoacidosis may increase the risk of hypoglycemia.An algorithm for insulin adjustment would be bene cial when combining SGLT2 inhibitor treatment with insulin in patients with type 1 diabetes mellitus.We therefore aim to explore whether the reduction of the basal insulin dose combined with SGLT2 inhibitor in patients with T1DM can reduce the frequency of hypoglycemia.We hypothesize that with an adequate basal insulin dose, the frequency of hypoglycemia will be higher if the basal insulin dose is not reduced when combined with the SGLT2 inhibitor.

Study protocol
The study is a multi-center, open-label, non-randomized, exploratory, prospective, interventional study (Figure 1).The subjects are strati ed into two groups based on the ratio of basal insulin (Basal) to the total daily insulin dose (TDD) (Basal/TDD, < 0.4 or >, 0.4).Instructions to the study subjects are found in Table 1.
A pre-observation period of 4 weeks is set before the intervention (administration of SGLT2 inhibitor).During this period, study subjects will measure and record fasting plasma beta-hydroxybutyric acid (BOHB), ash glucose monitoring (FGM), and self-monitoring of blood glucose (SMBG).The start of SGLT2 inhibitor administration is set as day zero of the observation period.The study subjects will visit the research institutions 4 times; at weeks -4, 0, 2, and 4. The study subjects were given a digital camera and instructed to take a picture of each meal they eat throughout the observation period.
[Group A] Study subjects reduce basal insulin dose by 0% (instructed to reduce bolus insulin dose) Subjects whose Basal/TDD ratio is < 0.4 are instructed not to reduce basal insulin dose but to reduce bolus insulin dose by 10%.The 10% reduction of the bolus insulin dose is based on 90% carbohydrate insulin ratio (CIR), and is rounded to the nearest whole number in case of insulin therapy with multiple daily injections (MDI), or rounded down to two decimal places in case of continuous subcutaneous insulin infusion (CSII).The study subjects are instructed to follow this instruction for 3 days from the start of the intervention.After the fourth day, subjects can titrate both basal and bolus dose according to "Algorithm for Basal Insulin Titration after SGLT2 administration (Figure 2)" and "Algorithm for Bolus Insulin Titration after SGLT2 administration (Figure 3)." [Group B] The study subjects are instructed to reduce basal insulin by 10% Study subjects whose Basal/TDD is >0.4 are instructed to reduce total insulin dose by 10%, reducing the basal insulin dose only.The dose of basal insulin is rounded down to one decimal places in case of MDI or to two decimal places in case of CSII.The study subjects are instructed to follow this instruction for 3 days from the start of the intervention.After the fourth day, subjects can titrate both basal and bolus dose according to "Algorithm for Basal Insulin Titration after SGLT2 administration (Figure 2)" and "Algorithm for Bolus Insulin Titration after SGLT2 administration (Figure 3)."

Eligibility Criteria
In line with the objectives of the study and for ensuring the safety of the subjects, the inclusion and exclusion criteria explicated in table 2 were set.In order to appropriately evaluate the e cacy of the study drugs, patients who require a legal representative are excluded.

Observations
Observations and the schedule are shown in table 3 and table 4. In principle, the study subjects will visit the research institutions, and at every visit, blood tests (fasting) and urine tests (spot) is performed.Investigators collect and enter the results of examinations listed in table 3 in the case report form (CRF), and send the CRF to the data center.Adverse events are followed as safety endpoints throughout the study.The items which the study subjects themselves measure are recorded onto the speci c documents and sent to the data center via the investigators.

Data management and monitoring
Data collection and management are carried out by third-party entities to avoid bias.The data management is performed by Soiken Inc.Data Management Group (the Data Centre).The Data Centre prepared a procedure manual for data management.The Data Centre's approval is required prior to sending any data related to the subjects in an electronic format.If data is transmitted over an unsecured electronic network, the data must be encoded at the source.Linkable anonymization by central registration number is used to identify the subjects.The investigators are responsible for appropriate storage of the correspondence table prepared by them to identify the subjects, in accordance with the procedures at the particular research institution.This correspondence table will be retained for 5 years after the completion of this study.Appropriate measures such as encoding or deletion are taken to ensure that the subjects cannot be identi ed, in accordance with applicable laws and regulations.
The Data Centre monitors this study to manage and ensure quality.The monitoring manager monitors the subjects in accordance with the prescribed manual on monitoring.For data quality management, the principal investigator and Central Committee con rms the progress of this study as necessary through the Data Centre to ensure conformance with the protocol and the Ethical Guidelines for Medical and Health Research Involving Human Subjects (28 February 2017; Ministry of Education, Culture, Sports, Science and Technology/Ministry of Health, Labor and Welfare) and the Clinical Trials Act (14 April 2017; Ministry of Education, Culture, Sports, Science and Technology/Ministry of Health, Labor and Welfare).

Primary endpoints
Frequency of hypoglycemia per day con rmed by self-monitoring of blood glucose (SMBG) during the intervention period (administration of SGLT2 inhibitor) (number of hypoglycemia/day).

Secondary endpoints 1. Frequency of ketosis before and after the intervention
Ketosis is de ned as plasma BOHB ≧ 600 μmol/L [11][12].Frequency of ketosis is de ned as the proportion of the number of days in which ketosis occurs in the study subjects during the pre-observation period and the observation period after the intervention.Change in plasma microRNA pro le.
Change in cardiac function estimated by ultrasonic echocardiography (UCG).

Rationale underlying the sample size
This study is designed as an exploratory study, and no prior studies have reported the frequency of hypoglycemia after the administration of SGLT2 inhibitor.We hypothesized that the frequency of hypoglycemia per day would increase after the administration of SGLT2 inhibitor, if the dose of basal insulin was not titrated.The frequency of hypoglycemia has been reported to be 7 ± 6 times/month when insulin glargine is used in patients with TIDM [13].The assumed increased hypoglycemia, and the required sample sizes are plotted in gure 4. When 26 patients were in one group, the % increase in hypoglycemia was more than 60%, which was deemed adequate.With an estimated dropout rate of 10%, the planned number of subjects (60 subjects, 30 in each group) is thought to have enough statistical power for an increased hypoglycemia frequency of more than 60% (11.2 ± 6 times/month) in Group A (Basal/TDD is < 0.4, the study subjects are instructed to reduce basal insulin dose 0% but instructed to reduce bolus insulin dose 10%) from 7 ± 6 times/month in Group B (Basal/TDD is 0.4 or higher, the study subjects are instructed to reduce basal insulin 10%).
This study is being conducted at 7 research institutions where a total of 350 patients with T1DM are being treated.From a previous survey, 30% of these patients are eligible to use SGLT2 inhibitors as per the Japanese labelled indications, meaning that approximately 100 patients could potentially use the SGLT2 inhibitor with insulin.Among the 100 patients, all patients who sign a written consent form are enrolled in the study, and strati ed into Group A (Basal/TDD is < 0.4, subjects instructed to reduce basal insulin dose 0% and bolus insulin dose by 10%) or Group B (Basal/TDD >0.4,subjects instructed to reduce basal insulin by 10%).The proportion of patients who will consent is assumed to be 80%, and the proportion of patients who meet the inclusion criteria and none of the exclusion criteria is assumed to be 80%.Under these conditions, the target number of 60 study subjects is feasible.

Basic analytical procedure
For primary and secondary endpoints, both full analysis set (FAS) and per protocol set (PPS) will be analyzed.The analyses will be conducted with a two-sided signi cance level of 5%.A biomedical statistic expert created a statistical analysis plan (SAP) separately, and speci ed the details of the statistical methods including handling data.The SAP was prepared before the database lock.
When amendment of the SAP is needed, the data handling committee will carefully consider the effect of the amendment on the study itself, the results of the study, the ethical concerns and scienti c validity of the effect.If the data handling committee judges that the amendment of the SAP is valid, the procedure, the content, and the result of the discussion is recorded.
Analysis of study subjects' background information Summary statistics will be calculated for background information of study subjects per group.
Frequencies and percentages of categories are shown for nominal variables.Summary statistics (number of cases, mean, standard deviation, minimum, median, and maximum values) will be calculated for continuous variables.For comparisons between the groups, Chi-square test will be used for nominal variables.If the proportion of the cells with expected frequency of less than 5 is 20% or more, Fisher's exact test will be used.For comparisons between groups for continuous variables, two-sample t-test or Wilcoxon rank sum test is used.
Analysis of the primary endpoint Summary statistics will be calculated for the number of hypoglycemic events per day (plasma glucose level de ned by SMBG <70 mg/dL) during the intervention period (from baseline to week 4) using FAS as main analysis set and PPS for sensibility analysis set.For comparisons between groups, two-sample ttest and analysis of covariance will be conducted, and differences between the groups and their 95% con dence intervals calculated.HbA1c, age, and frequency of hypoglycemia (<70 mg/dL, con rmed by SMBG) per day during the pre-observation period before the intervention (week -4 to baseline) will be used as covariates in the analysis of covariance.If the data distribution does not follow a normal distribution, summary statistics will be calculated after logarithmic transformation.

Analysis of the secondary endpoints
For the analysis of the secondary endpoint 1 (frequency of ketosis) and 2 (frequency of hypoglycemia detected by FGM), summary statistics of measurements and changes will be calculated using FAS during the pre-observation period before the intervention (week -4 to baseline) and the observation period after the intervention (baseline to week 4) in each group.For the measurements, two-sample t-test will be used for comparisons between groups, and for the change, one-sample t-test will be used for comparison in each group.If the data distribution does not follow a normal distribution, summary statistics will be calculated after logarithmic transformation.
For the analysis of the secondary endpoint 3, summary statistics will be calculated using FAS for measurements at each observation point and change in the measurements from baseline to each observation point after the intervention.For the measurement, two-sample t-test will be used for comparisons between groups, and for the change in the measurement, one-sample t-test will be used for comparison in each group.

Analysis of the safety endpoints
For the analysis of the safety endpoints, a table of all adverse events and diseases or the like will be created for each group using the safety analysis set, and comparisons performed between groups as necessary using Fisher's exact test.

Analysis of exploratory endpoints
For the analysis or correlation between change in dietary habit, dietary content, and nutrient intake and change in fasting plasma BOHB acid, Spearman rank correlation coe cient and its 95% con dence interval will be calculated, and evaluated for signi cance.The subjects have been instructed to take pictures of each of their meals using digital camera.The stored images will be uploaded to a cloud, and diabetologists will analyze the images according to the Standard Tables of Food Composition in Japan using a specialized application (Asken, Wit Co., Ltd.).The volumes and calories of carbohydrates, proteins, fats, and nutrient intakes will be calculated by the system referred to as "online nutritional evaluation."

Ethics and Dissemination
The protocol was approved by Kyoto Prefectural University of Medicine, Clinical Research Review Board (CRB5180001), and registered at jRCT (jRCTs051190114).
The ndings will provide knowledge about the reduction of the basal insulin dose combined with an SGLT2 inhibitor in patients with T1DM, which may reduce the frequency of hypoglycemia associated with the combination therapy.The results will be disseminated through presentations at appropriate conferences and meetings, and publications in peer-reviewed journals.
All parties involved in this study will endeavor to protect the personal information of study subjects.This study is being conducted in accordance with the Personal Information Protection Law, and other legislation/law and regulations.Unique information (initial, carte number) of study subjects is stored securely in the research institutions, and information that allows a person outside the research institution to identify the study subjects (such as name, address, telephone number, etc.) is not included in CRFs and registration database.
A central registration number is used when the data center enquires regarding data from a study subject at a research institution.Investigators use the correspondence tables to identify their research subjects (anonymization), which are managed by themselves.Investigators store the correspondence tables securely and keep them properly until at least 5 years from the day of the completion of the study.The person responsible for personal information management at each research institution shall be the responsible investigator unless otherwise speci ed.
The principal investigator, the responsible investigators, sub-investigators, and the heads of the research institutions agree to store the study-related information properly until at least 5 years from the completion date of the study.The responsible person in charge of data management also stores the study-related information such as CRF as source documents and electronic information, such as data sets, are properly stored in the same way.
After the storage period, the data is to be discarded.Anonymized data collected for the analysis is kept stored for future secondary study such as meta-analysis.If the anonymized data is used for other studies, approval from the ethics review board is required before study implementation.
Samples for special blood tests are measured in laboratories utilized by each research institution.After the requisite data is obtained, the samples are disposed of, in accordance with standard procedural guidelines.
The study explores whether the reduction of the basal insulin dose combined with SGLT2 inhibitor in patients with type 1 diabetes mellitus can reduce the frequency of hypoglycemia with an overall acceptable safety pro le.
The treatment intervention in this study is based on the Basal/TDD at consent.Therefore, the study subjects cannot always receive their desired treatment.However, all study subjects will receive regular medication, and will measure ketone body and blood glucose, and titrate both basal and bolus insulin after the fourth day of the intervention.As a result, the study subjects may receive better medical care than would be the case if they did not participate in this study.
To reduce the burden on study subjects, the change of medications during the study are not restricted.Special blood tests are conducted using the residual samples to reduce the burden caused by multiple blood draws.Therefore, generally, special compensation is not provided even when health damages are considered to be caused by the drug(s) used in the study.For reporting purposes, all incidences during the study will be treated similarly to health damage or medical accidents caused by regular medication.The compensation in such a case is based on the Adverse Drug Reaction Relief System of the Pharmaceutical and Medical Devices Agency, Product Liability Law, or product liability insurance.The principle investigator contracted the insurance for clinical study corresponding to any incidences.The current research is carried out in accordance with the Clinical Trial Act and other laws and regulations.Burdens and predicted risks are minimized as much as possible from the perspective of protecting human subjects.In particular, we believe that the risk of adverse effects can be minimized by discontinuing the study treatment as soon as a symptom emerges and providing treatments using appropriate procedures.
Based on the overall evaluation, and taking into account conceivable bene ts from the study participation, this study protocol is considered appropriate to implement and adequate in terms of ethical considerations.

Discussion
This study will clarify the e cacy and safety of SGLT2 inhibitor treatment in patients with type 1 diabetes mellitus (T1DM).
The dose of insulin should be self-titrated based on the quantity of carbohydrate ingested.Therefore, therapy with only insulin may cause increased body weight, especially in T1DM patients with excess carbohydrate intake, which increases the risk of macrovascular complications.On the contrary, although low-carbohydrate diets can reduce the total insulin dose and the number of self-injections, it could result in nutritional imbalances.Increased mean body mass index (BMI) reported in T1DM patients [3] additionally increases the risk of cardiovascular disorders.
Recently, a SGLT2 inhibitor, dapagli ozin, was approved for T1DM when combined with insulin therapy.
SGLT2 inhibitors cause hypoglycemia less frequently, and leads to body weight loss in patients with type 2 diabetes mellitus [5].In addition, since SGLT2 inhibitors prevents cardiovascular complications in patients with type 2 diabetes mellitus [6], the bene cial effects of the SGLT2 inhibitors are also expected in patients with T1DM [7].
Hypoglycemia is one of the important factors that affects glycemic control in the treatment of T1DM.While addition of SGLT2 inhibitors might reduce the risk of hypoglycemia, their use has been reported to increase the frequency of diabetic ketoacidosis [8].Therefore, reduction of insulin dose to prevent hypoglycemia during adjunctive SGLT2 inhibitor treatment may increase the risk of ketoacidosis whereas maintained insulin dose in this setting to prevent ketoacidosis may increase the risk of hypoglycemia.The study subjects use the Freestyle Libre for FGM after receiving full explanation of how to use it.Since the sensor of the Freestyle Libre can be used for 2 weeks (14 days), investigators give 2 sensors to each study subject in advance of each period.
The study subject change and install sensor every 2 weeks.
The data which the study subjects measure by themselves are recorded onto the study subjects' diary.

Observation item
Fasting plasma beta-hydroxybutyric acid (ketone body) The study subjects measure ketone body once daily before the breakfast at home using Freestyle Libre (Abbott Japan Co., Ltd.)The study subjects also measure the ketone body at their free will at physical deconditioning, sick day or onset of symptoms suggesting ketoacidosis.

Plasma glucose (FGM)
The study subjects measure the plasma glucose continuously at home using Freestyle Libre (Abbott).
Plasma Glucose (SMBG) The study subjects conduct SMBG 4 times per day (before breakfast, before lunch, before dinner, and before bedtime) and at the awareness of hypoglycemic symptoms or at FGM values of less than 70 mg/dL at home using Freestyle (Abbott).

Dose of insulin
Awareness of hypoglycemia 12.Other items the study subjects measure by themselves 2 Observation point throughout observation period

Observation item diet and relevant information
The study subjects record the diet and relevant information every day throughout the observation period using the application software.
1) gender, age, height, target body weight, activities of daily life: prespeci ed value (enter once) 2) body weight, percent body fat, body water, alcohol intake, confectionery intake, staple food intake, main dish intake, side dish intake, milk product intake, fruit intake, number of steps (every day) 3) energy, protein, lipid, carbohydrate, dietary ber, sugar (every meal)

Observation item
Fasting plasma beta-hydroxybutyric acid (ketone body) The study subjects measure ketone body once daily before the breakfast at home using Freestyle Libre (Abbott Japan Co., Ltd.)The study subjects also measure the ketone body at their free will at physical deconditioning, sick day or onset of symptoms suggesting ketoacidosis.

Plasma glucose (FGM)
The study subjects measure the plasma glucose continuously at home using Freestyle Libre (Abbott).
Plasma Glucose (SMBG) The study subjects conduct SMBG 4 times per day (before breakfast, before lunch, before dinner, and before bedtime) and at the awareness of hypoglycemic symptoms or at FGM values of less than 70 mg/dL at home using Freestyle (Abbott).

Dose of insulin
Awareness of hypoglycemia 13. adverse event and disease or the like Observation point throughout observation period

Observation item
Classi cation, outcome, severity, relationship, etc. of adverse event and disease or the like.
Adverse event and disease or the like are observed throughout the study.Adverse event and disease or the like include the side effects of the medication and clinically signi cant abnormal uctuations in test results.Investigators collect the information of presence/absence of hypoglycemia, hypoglycemia, other adverse events and disease or the like by interview at every observation point.The occurrence of the adverse events and disease or the like is recorded onto the carte and CRF.The study subjects are further followed up if necessary.
*The classi cation of adverse events and disease or the like is based on MedDRA/J.
Refer "10.Safety evaluation analysis" The
study design The study subjects are strati ed into two groups based on the ratio of basal insulin (Basal) to the total daily insulin dose (TDD) (Basal/TDD, < 0.4 or >0.4).The study does not involve randomization of participants.

Table 3 .
Observation items