Original. Hirotaka Watada 1), Makoto Imori 2), Pengfei Li 3) and Noriyuki Iwamoto 2)

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1 2017, 64 (7), Original Insulin lispro 25/75 and insulin lispro 50/50 as starter insulin in Japanese patients with type 2 diabetes: subanalysis of the CLASSIFY randomized trial Hirotaka Watada 1), Makoto Imori 2), Pengfei Li 3) and Noriyuki Iwamoto 2) 1) Department of Metabolism and Endocrinology, Juntendo University Graduate School of Medicine, Tokyo, Japan 2) Medicines Development Unit Japan, Eli Lilly Japan K.K., Kobe, Japan 3) Medical Department, Lilly Suzhou Pharmaceutical Co. Ltd, Shanghai, China Abstract. In Japan, premixed insulins are commonly used as starter insulin for type 2 diabetes. This subpopulation analysis assessed the efficacy and safety of twice-daily LM25 (25% insulin lispro/75% insulin lispro protamine) and LM50 (50% insulin lispro/50% insulin lispro protamine) as starter insulin in Japanese subjects, and compared these results with the whole-trial populations of East Asian subjects. In this subpopulation analysis of an open-label, phase 4, randomized trial (CLASSIFY), Japanese subjects received LM25 (n = 88) or LM50 (n = 84) twice-daily for 26 weeks. The primary outcome was change from baseline at Week 26 in glycated hemoglobin (HbA1c). Results for Japanese subjects were generally similar to those for the whole-trial population. Similar changes from baseline in HbA1c were observed for LM25 and LM50 groups (least squares [LS] mean difference [95% confidence interval] of LM25 LM50 = 0.13 [-0.16, 0.41]%, 1.42 [-1.75, 4.48] mmol/mol, p = 0.388). More LM50-treated subjects than LM25-treated subjects achieved HbA1c targets of <7.0% (59.5% versus 43.2%; p = 0.034) or 6.5% (45.2% versus 28.4%; p = 0.027). The reduction in postprandial blood glucose concentrations after morning and evening meals was statistically significantly greater for LM50 than for LM25. The incidence of both hypoglycemia and treatment-emergent adverse events were similar between treatment groups. Both LM25 and LM50 twice daily appear to be effective and well tolerated as starter insulin, although LM50 might be more effective for Japanese type 2 diabetes patients. Key words: Insulin lispro, Japan, Premixed insulin ASIA HAS BECOME increasingly important as a source of new cases of type 2 diabetes, which represents a major global cause of morbidity and premature death [1]. In Japan, more than 7 million people have diabetes, which is mostly type 2 diabetes, and the World Health Organization predicts that diabetes will affect 8.9 million Japanese people by 2030 [2, 3]. Further, a population-based prospective study has shown that the prevalence of type 2 diabetes in Japan has increased substantially since the 1980s, most likely reflecting an increase in the prevalence of obesity and declining physical activity levels [4]. Submitted Jan. 24, 2017; Accepted Mar. 24, 2017 as EJ Released online in J-STAGE as advance publication May 24, 2017 Correspondence to: Makoto Imori, M.D., Medicines Development Unit Japan, Eli Lilly Japan K.K., Sannomiya Plaza Bldg Isogami-dori, Chuo-ku, Kobe , Japan. imori_makoto@lilly.com ClinicalTrials.gov identifier: NCT ; Funding: Eli Lilly. The Japan Endocrine Society The European Association for the Study of Diabetes and the American Diabetes Association jointly recommended in their 2015 position statement that basal insulin is the most appropriate starter insulin after failure of intensive oral antihyperglycemic medication (OAM) therapy in patients with type 2 diabetes [5]. However, premixed insulins are more commonly used in Japan as starter insulin than in Western countries [6]. This is most likely related to benefits of premixed insulin compared with basal insulin, including improved overall and postprandial glycemic control [7-9], as well as greater ease of therapy associated with reduced regimen complexity and injection frequency [10, 11]. The underlying need to take advantage of these benefits of premixed insulin in Japanese patients may relate to early deterioration of β-cell function leading to reduced insulin secretion [12, 13].

2 706 Watada et al. In Japanese populations, limited data suggest that mid-mix insulin (consisting of equal parts of rapid- and long-acting insulin) may provide better control of glycated hemoglobin (HbA1c), postprandial glucose (PPG), and circadian variation of blood glucose (BG) than low-mix insulin (consisting of a lower proportion of rapid-acting insulin compared with long-acting insulin) [14, 15]. However, further evidence is needed in Japanese patients to (i) support the use of mid-mix insulin as starter insulin, and (ii) guide physicians regarding the most appropriate choice of starter insulin for specific patients. The CLASSIFY randomized study in East Asian subjects found that low-mix premixed 25% insulin lispro/75% insulin lispro protamine (LM25; Humalog Mix 25) and mid-mix premixed 50% insulin lispro/50% insulin lispro protamine (LM50; Humalog Mix 50) provided similar glycemic control and tolerability over 26 weeks [16]. However, twice-daily LM50 appeared more effective than twice-daily LM25 for controlling morning and evening PPG and reducing HbA1c for those with higher baseline HbA1c or PPG, or with a high carbohydrate diet. LM50 also appeared to produce less nocturnal hypoglycemia than LM25. Although Japanese subjects also made up approximately 40% of the population of the CLASSIFY study, there is a need to ensure that results for the Japanese subpopulation match those seen for the whole-trial population of the primary randomized study. The main objective of this subpopulation analysis of the CLASSIFY study was to assess the efficacy and safety profiles of LM25 and LM50 as starter insulin in Japanese subjects and to compare these results with those of East Asian subjects (whole-trial population). Materials and Methods Study design This subpopulation analysis examined Japanese subjects with type 2 diabetes inadequately controlled with OAM enrolled in a phase 4, open-label, 26-week, parallel-arm, multinational, randomized trial that compared treatment with LM25 and LM50 (CLASSIFY study) [16]. The study included a 2- to 4-week screening/lead-in period followed by an intensive 12-week dose-adjustment period and, finally, a 14-week maintenance period. Of the 38 sites at which the study was conducted, 19 sites in Japan enrolled patients. The study was conducted according to the Declaration of Helsinki and International Conference of Harmonisation Good Clinical Practice. Relevant institutional review boards approved the study, which gained written informed consent from all subjects before enrollment. The primary randomized study was registered at (ClinicalTrials. gov identifier: NCT ). Study population This subpopulation analysis included subjects enrolled at sites in Japan. Japanese subjects were enrolled if they were aged 20 years with a diagnosis of type 2 diabetes 6 months and had received α-glucosidase inhibitors, biguanides, dipeptidyl peptidase IV inhibitor, glinide, sulfonylureas, or thiazolidinedione (TZD), or any of these OAMs in combination. Subjects also had to have stable OAM doses for 8 weeks (12 weeks for TZD) before screening (Visit 1), a body mass index (BMI) of 18.5 to 35.0 kg/m 2, and a screening HbA1c level 7.0% (53 mmol/mol) and 11.0% (97 mmol/mol). Exclusion criteria have been described previously [16]. Briefly, these included a diagnosis of acute myocardial infarction, cerebrovascular accident (stroke) 3 months before Visit 1, New York Heart Association class III or IV heart failure, current or previous insulin treatment (>7 days continuously), >1 episode of severe hypoglycemia 6 months before Visit 1, or type 1 diabetes. Patients with evidence of severe liver or renal disease were also excluded. Randomization and masking Methods of randomization and masking have been described in detail previously [16]. Briefly, randomization was performed via a computer-generated random sequence incorporating an interactive voice-response system with stratification according to HbA1c ( 8.5% or >8.5%), BG excursion ( 5 mmol/l or >5 mmol/l; based on the average of 2 self-monitored BG [SMBG] excursion measurements [difference between before breakfast and after breakfast BG] during the week before randomization), and country to ensure between-group comparability. Although treatment allocation was open label, investigators and the sponsor were blinded to this until final database lock. Treatment protocol Eligible patients were randomly assigned (1:1) to receive subcutaneous LM25 or LM50 twice daily

3 Starter premixed insulin lispro in Japan 707 (5 units within 15 minutes before breakfast and 5 units within 15 minutes before dinner). Doses were adjusted each week for up to 12 weeks in line with BG concentrations as previously described [16]. Target BG levels (mean SMBG of the previous 3 days) were >3.9 mmol/l and 6.1 mmol/l before breakfast and dinner. Baseline OAMs were continued throughout the enrollment phase but discontinued at Week 0 (Visit 3), except for biguanides and thiazolidinediones, which were continued throughout the 26-week treatment phase. Outcome measures The primary efficacy outcome measure was the least squares (LS) mean (95% confidence interval [CI]) change from baseline in HbA1c at 26 weeks. Secondary efficacy outcome measures detailed for this subpopulation analysis were the percentage of subjects achieving HbA1c of <7% (53 mmol/mol) or 6.5% (48 mmol/mol) at 26 weeks (overall, without hypoglycemia, without nocturnal hypoglycemia, without body weight gain, without weight gain and without hypoglycemia, and without weight gain and without nocturnal hypoglycemia), change from baseline in fasting BG (FBG), change in 1,5 anhydroglucitol (1,5-AG) concentration from baseline, 7-point SMBG profile (before and 2 hours after meals, plus bedtime), and BG excursion by meal (for each of breakfast, lunch, and dinner). The change in the total daily dose (TDD) of insulin was also assessed. The incidence (proportion of patients) and frequency (rate per patient-year exposure) of total, severe, and nocturnal hypoglycemic episodes were considered as a specific outcome measure of this subpopulation analysis. Hypoglycemia was defined as BG 3.9 mmol/l with or without symptoms, or symptoms of hypoglycemia even in the absence of confirmed BG reading. Severe hypoglycemia was defined as episodes of glycemia that required assistance. Subgroup analyses examined the potential differences in treatment effects on the change from baseline in HbA1c with lower and higher than median levels of baseline HbA1c, PPG, and FBG. Safety outcome measures included in this subpopulation analysis were the incidence of adverse events (including serious adverse events [AEs], and AEs leading to discontinuation), change from baseline in body weight, and injection site reactions / hypersensitivity (AEs involving antibody formation). Statistical analysis The sample size calculation method, as described previously [16], assumed a common standard deviation (SD) of 1.1%, a two-sided alpha level of 0.05, a 10% drop-out rate, and a noninferiority margin of 0.4%. The whole trial population study had a greater than 99% probability of reaching a conclusive outcome using the classification method [17]. However, no formal sample size consideration was made on the Japanese subgroup in the design stage of the study. The primary efficacy and safety analyses used the intention-to-treat population (all randomized subjects receiving 1 study drug dose) according to assigned treatment. Treatment differences were assessed using the LS mean change from baseline via a mixed-effects model with repeated measures. Fixed effects were BG excursion ( 90 mg/ dl or >90 mg/dl), pretreatment (α-glucosidase inhibitor, sulfonylureas, glinide, or dipeptidyl peptidase IV inhibitor) or not, treatment (LM25 or LM50), treatment-by-visit interaction, and visit. The random effect was patient, with baseline HbA1c acting as the covariate. Predefined subgroup analyses assessed the treatment effects of lower and higher than median baseline HbA1c, PPG, and FBG levels on the change in HbA1c from baseline. Analyses were performed using SAS Version 9.3 (Cary, NC, USA). Results Demographic and baseline clinical characteristics The Japanese subpopulation comprised 172 of the 403 randomized subjects from the whole-trial population. Study completion rates were higher in the Japanese subpopulation than in the whole-trial population for both the LM25 (98.9% vs 92.3%) and LM50 (94.0% vs 90.3%) groups (Supplementary Fig. 1). The baseline characteristics of subjects were generally similar between the Japanese subpopulation and whole-trial population for both treatment groups (Table 1). Similar to the whole-trial population, most subjects in the Japanese subpopulation were male and had long-standing, poorly controlled diabetes. However, mean HbA1c levels in the Japanese subpopulation (8.42% [68.53 mmol/mol]) were slightly lower than for the whole-trial population (8.56% [70.06 mmol/mol]). A higher proportion of patients in the Japanese subpopulation (37.2%) reported no previous OAM use compared with that of the whole-trial population (26.6%).

4 708 Watada et al. Glycemic control In this Japanese subpopulation, improvements in the glycemic control as expressed by the LS mean changes from baseline in HbA1c were similar for the LM25 and LM50 groups (Fig. 1). The LS mean (95% CI) change from baseline to 26 weeks in HbA1c was numerically greater in the LM50 group (-1.52 [-1.73, -1.32]%, [-18.91, ] mmol/mol) than in the LM25 group (-1.40 [-1.60, -1.20]%, [-17.49, ] mmol/mol), although the difference between treatment groups was not statistically significant (LS mean difference LM25 LM50 = 0.13 [-0.16, 0.41]%, 1.42 [-1.75, 4.48] mmol/mol, p = 0.388). The mean (SD) HbA1c level at endpoint was slightly lower in the LM50 group (6.81 [0.83]%, [9.07] mmol/ mol) compared with the LM25 group (7.07 [0.81]%, [8.85] mmol/mol). The results of the primary efficacy measure were similar for the Japanese subpopulation and the whole-trial population (LS mean difference LM25 LM50 for whole-trial population = 0.17 [- 0.01, 0.35]%, 1.86 [-0.11, 3.83] mmol/mol, p = 0.072). Table 1 Baseline characteristics of subjects with type 2 diabetes treated with LM25 and LM50 for whole-trial population and Japanese subpopulation Whole-trial population Japanese subpopulation Variable LM25 (N = 207) LM50 (N = 196) LM25 (N = 88) LM50 (N = 84) Age, mean years (SD) 55.7 (10.0) 57.3 (9.7) 54.1 (10.8) 56.3 (10.4) Female, n (%) 76 (36.7) 77 (39.3) 19 (21.6) 25 (29.8) Height, mean cm (SD) (9.4) (8.8) (9.6) (8.9) Weight, mean kg (SD) 69.4 (12.2) 69.5 (12.8) 73.9 (12.7) 71.9 (12.1) BMI, mean kg/m 2 (SD) 25.7 (3.2) 25.7 (3.8) 26.7 (3.3) 26.4 (3.5) Race, n (%) Asian 192 (92.8) 182 (92.9) 88 (100.0) 84 (100.0) HbA1c, mean % (SD) 8.60 (1.04) 8.52 (1.15) 8.49 (0.97) 8.35 (1.14) Disease duration, mean years (SD) 9.7 (6.2) 9.2 (6.4) 9.2 (5.4) 9.8 (6.8) OAM use, n (%) None 54 (26.1) 53 (27.0) 30 (34.1) 34 (40.5) Biguanide only 134 (64.7) 125 (63.8) 48 (54.5) 39 (46.4) TZD only 8 (3.9) 9 (4.6) 5 (5.7) 6 (7.1) Biguanide + TZD 11 (5.3) 9 (4.6) 5 (5.7) 5 (6.0) Abbreviations: BMI, body mass index; HbA1c, glycated hemoglobin; LM25, 25% insulin lispro/75% insulin lispro protamine; LM50, 50% insulin lispro/50% insulin lispro protamine; OAM, oral antihyperglycemic medication; n, number; N, total number; SD, standard deviation; TZD, thiazolidinedione. Fig. 1 Time course of mean (± standard deviation [SD]) change in HbA1c levels during the 26-week study period for the Japanese subpopulation HbA1c, glycated hemoglobin; LM25, 25% insulin lispro/75% insulin lispro protamine; LM50, 50% insulin lispro/50% insulin lispro protamine.

5 Starter premixed insulin lispro in Japan 709 Overall, a significantly higher percentage of Japanese subjects in the LM50 group compared with the LM25 group achieved a target HbA1c <7% (53 mmol/mol) or 6.5% (48 mmol/mol) at 26 weeks, which corresponds with the results for the whole-trial population (Fig. 2). A higher proportion of Japanese subjects in the LM50 group compared with the LM25 group achieved both HbA1c targets at 26 weeks without weight gain or without nocturnal hypoglycemia (Supplementary Table 1). However, unlike in the whole-trial population, differences between treatment groups for these comparisons were not statistically significant. The LS mean (95% CI) changes in FBG from baseline were similar for the LM25 and LM50 groups of the Japanese subpopulation (Supplementary Table 2). The difference in the LS mean (95% CI) change in FBG between the LM25 and LM50 groups was not statistically significant (-0.25 [-0.79, 0.30] mmol/l, p = 0.371). In agreement with this finding, there was essentially no difference in the change over time in FBG from baseline to 26 weeks for the LM25 and LM50 groups (Supplementary Fig. 2). A similar trend was seen for the LS mean change in 1,5-AG from baseline to 26 weeks. The difference in the LS mean change in 1,5-AG from baseline to 26 weeks between the LM25 and LM50 groups was also not statistically significant (-0.96 [-2.26, 0.34] µg/ml, p = 0.145) (Supplementary Table 2). Further, the changes over time in 1,5-AG from baseline to 26 weeks were similar for the LM25 and LM50 groups (Supplementary Fig. 3). These results were generally similar to those observed for the whole-trial population [16]. Postprandial glucose control Structured 7-point SMBG profiles showed that the PPG control improved at Week 26 to a significantly greater extent in the LM50 group than in the LM25 group after the morning (p = 0.036) and evening (p = 0.014) meals (Fig. 3, Supplementary Table 3). Further, the LS mean SMBG change from baseline in the LM50 group was significantly greater than in the LM25 group for morning (p = 0.001) and evening (p = 0.031) meals. The LS mean (95% CI) change in average SMBG excursion was (-0.94, 0.24) mmol/l for the LM25 group and (-1.73, -0.99) mmol/l for the LM50 group (mean difference = 0.77 [0.29, 1.26] mmol/l; p = 0.002; Supplementary Fig. 4). Similar results to those of the Japanese subpopulation were observed in the whole-trial population for both the Week 26 structured 7-point SMBG profiles and the LS mean change in SMBG from baseline for each meal excursion [16]. Fig. 2 Percentage of subjects achieving target HbA1c levels of <7% (<53 mmol/mol) (left panel) or 6.5% ( 48 mmol/mol) (right panel) at 26 weeks for the whole-trial population and Japanese subpopulation HbA1c, glycated hemoglobin; LM25, 25% insulin lispro/75% insulin lispro protamine; LM50, 50% insulin lispro/50% insulin lispro protamine. Fig. 3 Seven-point self-monitored blood glucose (SMBG) profiles in Japanese subjects with type 2 diabetes treated with 25% insulin lispro/75% insulin lispro protamine (LM25) or 50% insulin lispro/50% insulin lispro protamine (LM50) at baseline and endpoint (26 weeks) p values based on analysis of covariance of the change from baseline to endpoint between LM25 and LM50 groups. * p = 0.036, *** p =

6 710 Watada et al. Hypoglycemia The overall incidence of hypoglycemic episodes, including all episodes and nocturnal episodes, was highly similar between the LM25 and LM50 groups. Overall, 285 hypoglycemic episodes occurred in 61 subjects (69.3%) in the LM25 group compared with 352 episodes in 61 subjects (72.6%) in the LM50 group. Further, 28 nocturnal episodes occurred in 9 subjects (10.2%) in the LM25 group compared with 18 episodes in 11 subjects (13.1%) in the LM50 group. The frequencies of these different types of hypoglycemic episodes were highly similar between the LM25 and LM50 groups (Table 2). The frequency of hypoglycemic episodes in the Japanese and all-trial populations were very similar. No episodes of severe hypoglycemia were recorded at any visit. Total daily dose of insulin The TDD of insulin increased similarly over the first 12 weeks in the LM25 and LM50 groups. The TDD of insulin remained relatively constant after 12 weeks and, at 26 weeks, reached a mean daily dose (SD) of 41.7 (19.6) IU in the LM25 group and 39.5 (18.9) IU in the LM50 group [16]. Predefined subgroup analyses Japanese subjects treated with LM50 had numerically greater changes from baseline in HbA1c than those treated with LM25 for subjects with (i) baseline HbA1c at or above the median, (ii) baseline PPG at or above the median, (iii) baseline FBG at or above median, or (iv) carbohydrate intake (as percentage total daily energy intake) at or above the median (Supplementary Table 4). These results were similar to those seen for the whole-trial population, although the differences were not statistically significant in the Japanese subpopulation. Change from baseline in HbA1c was not different between treatment groups for Japanese subjects with lower than median baseline HbA1c, PPG, FBG, or carbohydrate intake subgroups (Supplementary Table 4). A similar result was also noted in the whole-trial population [16, 18]. Safety and tolerability measures In the Japanese subpopulation, LM25 and LM50 were both well tolerated, in agreement with the results for the whole-trial population. Similar overall rates of treatment-emergent AEs were seen with the LM25 (67.0%) and LM50 (71.4%) groups and no Japanese subjects discontinued because of an adverse event (Supplementary Fig. 1). Rates of AEs related to injection and hypersensitivity reactions involving antibody formation were very low. The mean change (SD) in body weight was similar for the LM25 group (2.67 [2.51] kg) and the LM50 group (2.51 [3.85] kg) (p = 0.922) [16]. Discussion This subpopulation analysis of an open-label, randomized clinical trial compared LM25 with LM50 given twice daily as starter insulin for the treatment of type 2 diabetes in Japanese subjects. Primarily, we found that glycemic control improved with both LM25 Table 2 Frequency of hypoglycemia (per person per year) during the 26-week study period for Japanese subjects with type 2 diabetes treated with LM25 and LM50 Hypoglycemic events, Event type per person per year (SD) p value LM25 LM50 Total (8.788) (9.975) Nocturnal (2.397) (1.573) Severe No events No events ND Abbreviations: LM25, 25% insulin lispro/75% insulin lispro protamine; LM50, 50% insulin lispro/50% insulin lispro protamine; ND, not determined; SD, standard deviation. Symptomatic or asymptomatic hypoglycemia with documented plasma glucose concentration 3.9 mmol/l, or probable symptomatic hypoglycemia without plasma glucose measurement. Hypoglycemic event occurring between bedtime and waking. Hypoglycemic event requiring assistance from another person to actively administer carbohydrate, glucagons, or other resuscitative actions.

7 Starter premixed insulin lispro in Japan 711 and LM50 in terms of HbA1c changes from baseline to 26 weeks, with a numerically larger but not statistically significant improvement in the LM50 group. Further, significantly greater percentages of Japanese subjects treated with LM50 achieved a target HbA1c <7% or 6.5% at 26 weeks than for those treated with LM25. Rates of hypoglycemia and nocturnal hypoglycemia for subjects treated with LM25 and LM50 were not statistically significantly different. Subgroup analyses found numerically greater changes from baseline in HbA1c in LM50-treated subjects, compared with LM25-treated subjects, in subjects with levels of several parameters at or above the median. However, these results lacked statistical significance observed for the whole-trial population. Finally, in accordance with the findings for the whole-trial population, this Japanese subpopulation analysis found statistically significantly lower post-breakfast and post-dinner BG levels in the LM50 group than in the LM25 group. Combining these observations, we conclude that LM50 might represent a more effective option as starter insulin for Japanese patients with type 2 diabetes than LM25. In general, the results from this Japanese subpopulation were similar to those from the whole-trial population [16], especially in terms of overall improvements in glycemic control, attainment of predefined HbA1c targets, and rates of hypoglycemic episodes. However, there are some key differences in the relative response to LM50 and LM25 in the Japanese and Chinese subpopulations that are worth considering. Most importantly, LM50 was found to be significantly more efficacious than LM25 in reducing mean HbA1c levels in the Chinese subpopulation, whereas this advantage did not achieve statistical significance in the Japanese subpopulation. Possible reasons for this relate to baseline differences between these two subpopulations. First, mean baseline HbA1c levels were higher in the Chinese subpopulation; patients with higher baseline HbA1c levels were shown to have a greater response to LM50 than patients with lower baseline HbA1c levels in the subgroup analysis of the Chinese subpopulation study and similar results were noted in the first year results of the Treating to Target in Type 2 Diabetes (4-T) study [19]. Second, there was a greater proportion of female patients in the Chinese than the Japanese subpopulation, which may be relevant to the difference in primary efficacy although the mechanism of this is not clear. Last, a statistically greater efficacy of LM50 over LM25 at reducing HbA1c was noted in Chinese patients with baseline HbA1c and postprandial glucose greater than or equal to median levels. In this regard, the overall phenomenon was also noted in Japanese patients albeit to a lesser extent, prompting the speculation that this ethnic difference might be caused by the different response of post-dinner blood glucose to premixed insulin judged from the SMBG profile in both Japanese and Chinese patients. Unlike the whole trial population, this Japanese substudy found that FBG levels were similar for both LM25 and LM50 groups, despite the lower percentage of intermediate-acting insulin lispro protamine in LM50. The most obvious possible reason for this is the smaller sample size in this Japanese subpopulation, which limited the statistical power to detect a treatment difference. Further, although intermediate-acting protamine insulin is expected to have a greater effect than on FBG, the impact of this is likely to be less apparent than the relatively greater impact of rapid-acting insulin on post-prandial glucose levels. We also noted that a high proportion (>50%) of patients in this Japanese subpopulation received biguanide therapy, which is known to reduce FBG and may have influenced the difference between treatment groups. The proportion of subjects achieving a target without nocturnal hypoglycemia differed numerically between the LM25 and LM50 groups, although the proportion of subjects achieving a target HbA1c level without hypoglycemia did not. However, asymptomatic nocturnal hypoglycemia was not assessed in this study. Further, the proportion of subjects achieving a target HbA1c level without weight gain was not significantly greater in the LM50 group of the Japanese subpopulation, whereas this was observed for the whole-trial population. A possible reason for this might relate to greater absolute and relative changes in body weight from baseline in the Japanese subpopulation compared with the whole-trial population. The observation of statistically significantly lower post-breakfast and post-dinner BG levels in the LM50 group compared with the LM25 group may have clinical significance for Japanese subjects with type 2 diabetes. Guidelines from the International Diabetes Federation strongly recommend targeting postprandial hyperglycemia, which is associated with serious complications across a range of HbA1c values [20]. Further, although postprandial hyperglycemia worsens with worsening diabetes [21], this makes its strongest relative contribution at near normal HbA1c levels.

8 712 Watada et al. A small number of other published studies have compared LM25 with LM50 or similar premixed insulin combinations. In a 12-week randomized study of insulin-naive Chinese patients with type 2 diabetes, LM50 twice daily for 12 weeks was associated with better glycemic control (HbA1c 5.5 ± 1.4% vs 6.5 ± 1.5%, p < 0.05) and more rapid achievement of target blood glucose levels (12.6 ± 3.6 days vs 22.3 ± 4.7 days, p < 0.05) than LM25 twice daily, without an increase in hypoglycemic episodes [22]. Another randomized clinical trial found that mid-mix biphasic insulin aspartate (BIAsp) three times daily also improved glycemic control to a greater extent than low-mix BIAsp twice daily without a causing a higher incidence of hypoglycemia [23]. In contrast, an open-label randomized study found that switching to LM50 from LM25 in 302 patients with type 2 diabetes who had not achieved adequate glycemic control did not clearly produce an improvement in overall glycemic control in terms of endpoints similar to those used in the current study [24]. Overall, however, the results from this Japanese subpopulation are supported by similar studies in the existing literature. We acknowledge that other treatment options may be applicable in patients requiring starter insulin, although the results of this study do not allow for direct comparisons. Basal supported oral therapy is also recommended by international and Japanese treatment guidelines for patients who require addition of insulin [5, 25]. Premixed insulin, Humalog Mix 50, is also now approved for three times daily administration after initial twice daily therapy in Japan and may provide tighter postprandial control than twice daily administration, but this administration regimen is not approved as starter insulin. Finally, newer formulations of insulin such as IDegAsp that combine rapid-acting and long-acting insulin in a fixed dose ratio are now available that may offer certain advantages. However, ongoing formal assessment in clinical comparative studies is needed to assess their relative benefits against current premixed starter insulin regimens. This subpopulation analysis is based on a study that has several strengths and limitations. Key strengths of the primary study are the use of a regional population, randomized controlled trial design, and endpoints relevant to a real-world clinical setting whereas the key strength of this subpopulation analysis is the focus on Japanese subjects. A key limitation of the primary study is the use of an open-label design; other limitations include the lack of stratification (eg, by OAM class) upon randomization, and inadequate statistical power for subgroup analyses. Further, asymptomatic nocturnal hypoglycemia is now recognized as a common phenomenon in many patients with diabetes, but requires continuous glucose monitoring for detection. However, as our study relied on SMBG for detection of hypoglycemic events, we were not able to assess the possible impact of such asymptomatic events. In conclusion, both LM25 and LM50 twice-daily treatment appear to be effective as starter insulin in Japanese patients with type 2 diabetes. However, for several reasons including the beneficial effects on glycemic control and postprandial glucose excursions, LM50 might be more effective than LM25 for Japanese patients with type 2 diabetes. Indeed, some numerical advantages were observed in several subgroup analyses in the LM50 group compared with the LM25 group. However, the reduced sample size meant that it was not possible to demonstrate statistical significance for these differences. Finally, both LM25 and LM50 were well tolerated and had similar hypoglycemia-related safety profiles in Japanese subjects. Acknowledgments Funding support Medical writing assistance was provided by Mark Snape, MB BS, CMPP, and Rebecca Lew, PhD, CMPP of ProScribe Envision Pharma Group, and was funded by Eli Lilly & Company, manufacturer of Humalog. ProScribe s services complied with international guidelines for Good Publication Practice (GPP3). Role of the sponsor Eli Lilly & Company was involved in the study design, data collection, data analysis, and preparation of the manuscript. Role of the contributors All authors participated in the drafting, critical revision, and approval of the final version of the manuscript. HW, PL, and NI were involved in the study design and data collection. HW, MI, and NI were involved in the interpretation of study results, and PL conducted the statistical analysis. Conflicts of interest MI and NI are employees of Eli Lilly Japan K.K. PL is an employee of Lilly Suzhou Pharmaceutical Co. Ltd.

9 Starter premixed insulin lispro in Japan 713 HW serves as an advisory panel member for and/or has received lecture fees and/or research funds from Abbott Japan, Astellas Pharma Inc., AstraZeneca, Bayer, Benefit One Health Care Co. Ltd., Boehringer Ingelheim, Bristol-Myers Squibb, Daiichi Sankyo Inc., Dainippon Sumitomo Pharma, Eli Lilly Japan K.K., Johnson & Johnson, Kissei Pharmaceutical Co., Kowa Co., Kyowa Hokko Kirin Co., Mitsubishi-Tanabe Pharma, Mochida Pharmaceutical Co., Merck Sharp and Dohme, Nitto Boseki Co. Ltd., Novartis Pharmaceuticals, Novo Nordisk Pharma, Ono Pharmaceutical Co., Pfizer Inc., Sanofi, Sanwa Kagaku Kenkyusho Co., Shionogi Inc., Taisho Toyama Pharmaceutical Co., Takeda Pharmaceutical Co., and Terumo Corp. Other contributors / acknowledgments The authors would like to thank all study investigators and participants. Supplementary Fig. 1 Subject flow diagram Abbreviations: LM25, 25% insulin lispro/75% insulin lispro protamine; LM50, 50% insulin lispro/50% insulin lispro protamine; N, total number in group; n, number. Reasons for discontinuation for the whole-trial population are described in the CLASSIFY study [16]. Supplementary Fig. 2 Time course of fasting blood glucose (FBG) concentrations during the 26-week study period Shown are means ± standard deviation (SD). Abbreviations: LM25, 25% insulin lispro/75% insulin lispro protamine; LM50, 50% insulin lispro/50% insulin lispro protamine. Supplementary Fig. 3 Time course of 1,5-anhydroglucitol (1,5- AG) concentration during the 26-week study period Shown are means ± standard deviation (SD). Abbreviations: LM25, 25% insulin lispro/75% insulin lispro protamine; LM50, 50% insulin lispro/50% insulin lispro protamine.

10 714 Watada et al. Supplementary Fig. 4 Change from baseline in blood glucose (BG) excursions at 26 weeks in Japanese subjects with type 2 diabetes mellitus treated with 25% insulin lispro/75% insulin lispro protamine (LM25) or 50% insulin lispro/50% insulin lispro protamine (LM50) for the average self-monitored BG (SMBG) excursion. Supplementary Table 1 Subgroup analyses of the incidence of weight gain, total hypoglycemia, and/or nocturnal hypoglycemia in Japanese subjects with type 2 diabetes treated with LM25 and LM50 who achieved HbA1c targets of <7% (53 mmol/mol) or 6.5% (48 mmol/mol) Subgroup LM25 (N = 88) LM50 (N = 84) p value Achieved HbA1c <7% (<53 mmol/mol) No weight gain, n (%) 8 (9.1) 13 (15.5) No hypoglycemia, n (%) 14 (15.9) 12 (14.3) No nocturnal hypoglycemia, n (%) 34 (38.6) 45 (53.6) No weight gain and no hypoglycemia, n (%) 2 (2.3) 3 (3.6) No weight gain and no nocturnal hypoglycemia, n (%) 7 (8.0) 12 (14.3) Achieved HbA1c 6.5% ( 48 mmol/mol) No weight gain, n (%) 5 (5.7) 9 (10.7) No hypoglycemia, n (%) 8 (9.1) 7 (8.3) > No nocturnal hypoglycemia, n (%) 21 (23.9) 28 (33.3) No weight gain and no hypoglycemia, n (%) 0 (0.0) 2 (2.4) No weight gain and no nocturnal hypoglycemia, n (%) 5 (5.7) 8 (9.5) Abbreviations: HbA1c, glycated haemoglobin; LM25, 25% insulin lispro/75% insulin lispro protamine; LM50, 50% insulin lispro/50% insulin lispro protamine; N, total number in group; n, number. Supplementary Table 2 Secondary (FBG, 1,5-AG) efficacy measures for Japanese subjects with type 2 diabetes treated with LM25 and LM50 at 26 weeks Variable Treatment Baseline, mean (SD) Endpoint, mean (SD) LS mean change from baseline (95% CI) Difference (95% CI) in LS mean change from baseline p value FBG (mmol/l) LM (2.424) 7.23 (2.089) (-2.70, -1.93) LM (2.224) 7.36 (1.803) (-2.47, -1.66) (-0.79, 0.30) ,5-AG (μg/ml) LM (3.213) 7.48 (4.257) 2.71 (1.79, 3.64) LM (4.808) 9.32 (5.790) 3.67 (2.72, 4.63) (-2.26, 0.34) Number of subjects: LM25, n = 88; LM50, n = 84. Abbreviations: 1,5-AG, 1,5-anhydroglucitol; CI, confidence interval; FBG, fasting blood glucose; LM25, 25% insulin lispro/75% insulin lispro protamine; LM50, 50% insulin lispro/50% insulin lispro protamine; LS, least squares; SD, standard deviation.

11 Starter premixed insulin lispro in Japan 715 Supplementary Table 3 Seven-point self-monitored blood glucose profile at 26 weeks for Japanese subjects with type 2 diabetes treated with LM25 and LM50 Timepoint Treatment * LS Mean (95% CI) change from baseline, mmol/l Difference (95% CI) in LS mean change from baseline, mmol/l p value Pre-breakfast LM (-2.97, -2.28) LM (-2.51, -1.77) (-0.96, -0.00) Post-breakfast LM (-4.81, -3.71) LM (-5.63, -4.50) 0.81 (0.06, 1.55) Pre-lunch LM (-3.28, -2.48) LM (-3.49, -2.65) 0.19 (-0.36, 0.75) Post-lunch LM (-2.00, -0.86) LM (-2.12, -0.92) 0.09 (-0.70, 0.88) Pre-dinner LM (-2.20, -1.25) LM (-2.21, -1.21) (-0.68, 0.64) Post-dinner LM (-3.90, -2.78) LM (-4.93, -3.74) 1.00 (0.21, 1.78) Bedtime LM (-4.04, -3.01) LM (-4.62, -3.54) 0.56 (-0.16, 1.27) * Number of subjects: LM25, n = 88; LM50, n = 84. Abbreviations: LM25, 25% insulin lispro/75% insulin lispro protamine; LM50, 50% insulin lispro/50% insulin lispro protamine; LS, least squares. Supplementary Table 4 Subgroup analyses of the change from baseline in HbA1c for Japanese subjects with type 2 diabetes treated with LM25 and LM50, stratified by baseline HbA1c, baseline PPG, baseline FBG, daily carbohydrate intake, and carbohydrate as a percentage of total energy intake (below the median vs at or above the median) HbA1c, % Subgroup Treatment (n) Baseline Mean (SD) Endpoint Mean (SD) LS mean change from baseline (95% CI) Difference (95% CI) in LS mean change from baseline p value Baseline HbA1c 8.5% ( 69 mmol/mol) LM25 (54) 7.88 (0.413) 6.81 (0.649) (-1.23, ) LM50 (52) 7.61 (0.571) 6.61 (0.639) (-1.23, ) 0.00 (-0.26, 0.26) >8.5% (>69 mmol/mol) LM25 (34) 9.48 (0.759) 7.49 (0.870) (-2.35, -1.63) LM50 (32) 9.54 (0.742) 7.17 (1.012) (-2.74, -1.98) 0.37 (-0.15, 0.90) Baseline PPG <12.92 mmol/l LM25 (39) 8.07 (0.679) 6.97 (0.568) (-1.29, -0.77) LM50 (47) 7.79 (0.809) 6.68 (0.640) (-1.32, -0.84) 0.05 (-0.24, 0.34) mmol/l LM25 (49) 8.83 (1.037) 7.14 (0.957) (-1.99, -1.38) LM50 (37) 9.06 (1.107) 6.97 (1.009) (-2.40, -1.71) 0.37 (-0.09, 0.83) Baseline FBG <9.08 mmol/l LM25 (42) 7.97 (0.573) 6.84 (0.626) (-1.35, -0.90) LM50 (42) 7.80 (0.812) 6.74 (0.790) (-1.28, -0.82) (-0.39, 0.24) mmol/l LM25 (46) 8.97 (1.014) 7.28 (0.898) (-1.96, -1.37) LM50 (41) 8.94 (1.142) 6.86 (0.884) (-2.35, -1.73) 0.38 (-0.05, 0.80) Carbohydrate intake < g/day LM25 (43) 8.55 (0.997) 6.97 (0.793) (-1.85, -1.23) LM50 (43) 8.44 (1.317) 6.82 (0.808) (-1.96, -1.35) 0.12 (-0.31, 0.55) g/day LM25 (45) 8.44 (0.947) 7.16 (0.817) (-1.53, -1.01) LM50 (41) 8.25 (0.923) 6.79 (0.867) (-1.63, -1.07) 0.08 (-0.30, 0.46) Carbohydrate as percentage of total energy intake <54% LM25 (44) 8.57 (1.054) 7.13 (0.928) (-1.66, -1.09) LM50 (42) 8.25 (1.138) 6.84 (0.942) (-1.72, -1.14) 0.05 (-0.35, 0.46) % LM25 (44) 8.42 (0.879) 7.01 (0.665) (-1.69, -1.12) LM50 (42) 8.45 (1.145) 6.78 (0.704) (-1.91, -1.32) 0.21 (-0.20, 0.62) Abbreviations: CI, confidence interval; FBG, fasting blood glucose; HbA1c, glycated hemoglobin; LM25, 25% insulin lispro/75% insulin lispro protamine; LM50, 50% insulin lispro/50% insulin lispro protamine; LS, least squares; n, number; PPG, postprandial glucose; SD, standard deviation.

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