Pharmacokinetic and Pharmacodynamic Drug‐Drug Interactions Between Cofrogliptin and Metformin in Healthy Subjects: A Single‐Center, Single‐Arm, Phase I Study in China

Abstract

This single‐center, single‐arm, Phase I study evaluated drug–drug interactions (DDIs) between cofrogliptin and metformin in healthy Chinese subjects. Twenty‐two subjects sequentially received metformin monotherapy (Days 1–4), cofrogliptin monotherapy (Days 6–34), and combination therapy (Days 38–47). Serial blood/urine samples were collected for pharmacokinetic (PK) and pharmacodynamic (PD) analyses. Cofrogliptin showed no significant impact on metformin's PK and PD properties: the geometric mean ratios (GMRs, metformin + cofrogliptin/metformin) with 90% confidence intervals (CIs) for metformin AUC_0–8 h, AUC_0–12 h, C_{SS (max)}, Ae_0–8 h and Ratio_0–8 h, were 0.872 (0.809, 0.941), 0.900 (0.834, 0.971), 0.840 (0.743, 0.950), 0.899 (0.782, 1.032), and 0.994 (0.871, 1.136), respectively; 90% CIs for plasma glucose AUEC_0–4 h, AUEC_0–0.5 h, and EC_max of combination (combination vs cofrogliptin monotherapy) all fell within 0.80–1.25. Metformin did not statistically affect cofrogliptin's PK and PD properties, as the GMRs (metformin + cofrogliptin/cofrogliptin) (90% CIs) for cofrogliptin AUC over the dosing interval and C_{ss (max)} were 1.12 (1.072, 1.169) and 1.14 (1.057, 1.220), and AUEC_0–168 h, EC_max, and EC_min for dipeptidyl peptidase‐4 inhibition rate were 1.01 (1.003, 1.015), 1.00 (1.001, 1.009), and 1.03 (1.005, 1.040), respectively. No deaths, serious adverse events, or severe hypoglycemia occurred. In conclusion, the combination of cofrogliptin and metformin had no clinically significant PK or PD DDIs in healthy Chinese subjects.

Diabetes mellitus (DM) is a heterogeneous group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion and/or insulin action. ¹ DM has become one of the most prevalent chronic noncommunicable diseases that seriously threaten human health. ² Population growth, aging, urbanization, dietary habits, and lifestyle have led to a substantial increase in the DM population. The global prevalence of DM among individuals aged 20 to 79 years was estimated at 10.5% in 2021, with projections indicating an increase to 12.2% by 2045. ³ In China, the prevalence of DM is estimated to rise from 8.2% to 9.7% during 2020–2030. ⁴ Type 2 diabetes mellitus (T2DM) is the predominant form of DM, accounting for over 90% of global DM cases. ⁵ , ⁶ In China, T2DM affects approximately 141 million adults, corresponding to a prevalence of 12.4%. ⁷ Poorly controlled diabetes may lead to kidney failure, lower limb amputation, blindness, and various other long‐term health issues, all of which will compromise patients' quality of life. ⁸ Additionally, DM imposes a substantial economic burden on individuals, families, and healthcare system in many countries. ⁹ , ¹⁰

Currently, the treatment of T2DM includes insulin injection and oral hypoglycemic drugs. Metformin is a first‐line pharmacotherapeutic agent for the management of T2DM in clinical treatment guidelines, which primarily functions by regulating hepatic glucose production and peripheral insulin sensitivity. ¹¹ , ¹² , ¹³ However, after years of disease progression, poor adherence, suboptimal dosing, and comorbidities affect glucose metabolism, metformin monotherapy often fails to maintain satisfactory serum glucose levels. ¹⁴ Under these circumstances, clinicians often escalate therapy by titrating metformin dose or by combination therapy with second‐line antidiabetic agents to achieve optimal glycemic level. These agents include sulfonylureas, biguanides, glinides, thiazolidinediones, alpha‐glucosidase inhibitors, glucagon‐like peptide‐1 (GLP‐1) receptor agonists, and dipeptidyl peptidase‐4 (DPP‐4) inhibitors. ¹⁵ , ¹⁶ Among various antidiabetic drugs, the combination of DPP‐4 inhibitors is regarded as one of the optimal choices in terms of glucose‐lowering efficacy. DPP‐4 inhibitors have a low risk of hypoglycemia and other benefits, such as cardiorenal protection and weight reduction. ¹⁷ , ¹⁸

DPP‐4 inhibitors, as a useful class of oral hypoglycemic agents for treating T2DM, enhance glycemic control by augmenting the levels of active incretins. ¹⁹ Cofrogliptin (HSK7653), a novel, potent, ultralong‐acting oral DPP‐4 inhibitor, offers a promising therapeutic option for patients with T2DM through a dosing regimen of once every 2 weeks. ²⁰ , ²¹ Cofrogliptin plays a dual hormone regulatory role by inhibiting GLP‐1 degradation, increasing endogenous active GLP‐1 concentration, improving insulin levels, and inhibiting insulin glucagon section. ²² The primary components of cofrogliptin are structurally similar to omarigliptin, another long‐acting oral DPP‐4 inhibitor. At the same dosage of 30 mg/kg, the plasma DPP‐4 inhibition time of cofrogliptin was about four times as long as omarigliptin (72 vs 18 h). ²³ Several studies have reported that cofrogliptin has desirable glucose‐lowering effect with favorable safety and tolerability profiles in Chinese T2DM patients. ²⁰ , ²² , ²⁴

Drugs in combination prescriptions may interact with each another, thereby altering their pharmacokinetics (PK) and pharmacodynamics (PD) within human body. In fact, the combination therapy of DDP‐4 inhibitor and metformin has been proved to be effective and safe for T2DM treatment in clinical practice, with synergistic mechanisms to reduce patients' glycemic levels. ²⁵ , ²⁶ For example, combination therapy of evogliptin and metformin could effectively improve glycemic control in T2DM patients where metformin monotherapy did not work well, without clinically significant PK interactions. ²⁷ , ²⁸ As a DDP‐4 inhibitor with selective and sustained DPP‐4 inhibitory function, cofrogliptin is likely to be effective as an add‐on therapy to metformin. However, the potential drug–drug interaction (DDI) between cofrogliptin and metformin has not yet been investigated. The aim of the present study was to evaluate the PK and PD interactions of cofrogliptin and metformin in healthy Chinese subjects.

Methods

Study Design

This was a Phase I, single‐center, single‐arm study (Clinicaltrials.gov identifier: NCT06084156) designed to evaluate the PK interaction between cofrogliptin and metformin in healthy Chinese subjects. Secondary endpoints included PD and safety analyses. This study was performed in compliance with the Declaration of Helsinki and Good Clinical Practice guidelines, and was approved by the ethics committee of the Peking University Third Hospital (Institutional Review Board number: No. 2019‐060‐04). Written informed consent was obtained from all subjects prior to commencing the study. The study design comprised a 14‐day screening period, a 36‐h washout period, three distinct treatment periods, and a 7‐day follow‐up period (Figure 1). In treatment period 1 (metformin monotherapy, Day 1 to Day 4), subjects received oral metformin (Sino‐American Shanghai Squibb Pharmaceuticals Co. Ltd., Shanghai, China) 1000 mg twice daily (BID) followed by a 36‐h washout. In treatment period 2 (cofrogliptin monotherapy), subjects initially received cofrogliptin (Haisco Pharmaceutical group Co. Ltd., Chengdu, China) 35 mg once daily (QD) on Day 6, then received cofrogliptin 25 mg QD on Days 13, 20, 27, and 34. In treatment period 3 (combination therapy), cofrogliptin 25 mg QD was co‐administered with metformin 1000 mg BID from Day 38 to Day 47. The study drugs were administered with 250  mL of water in a fasting state after collecting PK and PD samples on Days 4, 27, and 41. On all other days, metformin was administered with meals, and cofrogliptin was given 1 h before meals.

Figure 1.

Study design schematic. BID, twice daily; DPP‐4, dipeptidyl peptidase‐4; OGTT, oral glucose tolerance test; PK, pharmacokinetics.

Subjects

This study recruited healthy Chinese subjects aged 18–60 years with a body mass index (BMI) of 18–28 kg/m² (weight ≥ 50 kg). Subjects who met any of the following criteria were excluded from this study: (1) those with any clinically significant medical abnormalities during physical examination, laboratory examination, 12‐lead electrocardiogram (12‐ECG), abdominal B‐ultrasonography, etc.; (2) fasting blood glucose <3.9 or ≥6.1 mmol/L; (3) any condition that could affect the absorption, distribution, metabolism, and excretion of the drug; (4) any positive screening result for serum hepatitis B surface antigen (HBsAg), hepatitis C antibody (HCVAb), treponema pallidum antibody (TP‐Ab), or human immunodeficiency virus (HIV); (5) treatment with an investigational drug within 3 months; and (6) any condition that the investigator considers inappropriate for study participation.

Pharmacokinetic Assay

Blood samples (2 mL) for determination of plasma concentration were collected at specified time points before or after the administration of cofrogliptin and metformin. For metformin, blood samples were collected pre‐dose on Days 1, 3, and 4, and 0.5, 1, 1.5, 2, 4, 6, 8, and 12 h post‐dose on Day 4. For cofrogliptin, blood samples were collected pre‐dose on Days 6, 13, 20, and 27, and 0.5, 1, 2, 4, 6, 8, 12, 24, 72, 120, and 168 h post‐dose on Day 27. During combination therapy, blood samples were collected pre‐dose on Days 40 and 41, and 0.5, 1, 2, 4, 6, 8, 12, 24, 72, 120, and 168 h post‐dose on Day 41. In addition, urine samples were collected pre‐dose on Day 1, and 12 h pre‐dose, and 0–2, 2–4, 4–8, and 8–12 post‐dose on Days 4 and 41 for PK analysis of metformin.

Plasma PK parameters included the area under the plasma concentration–time curve (AUC) from zero to infinity (AUC_inf) or during the dosing interval at steady state (AUC_tau), maximum plasma concentration (C_max), maximum steady‐state concentration (C_{ss (max)}) and minimum steady‐state concentration (C_{ss (min)}), average plasma concentration at steady state (C_{ss (avr)}), time to C_max (T_max), elimination rate constant (Kel), apparent terminal elimination half‐life (T_1/2), apparent clearance (CL/F), apparent volume of distribution (V_z/F), and renal clearance (CL_R). AUC was calculated by the linear trapezoidal log‐down rule. Urinary PK parameters of metformin included cumulative urinary excretion (Ae_0–t) and metformin/cumulative urinary excretion (Ae Ratio)_. Ae was calculated as concentration × volume. The CL_R was calculated as the ratio of Ae to plasma AUC within the same time interval.

Pharmacodynamic Assay

Blood samples (2 mL) were collected before medication on Day 6, before medication, and 2, 24, 72, 120, and 168 h after medication on Day 27 and Day 41 to determine DPP‐4 inhibition rate of cofrogliptin. DPP‐4 activity in plasma samples was measured using the DPP‐4 Activity Assay kit (Sigma). The PD parameters of DPP‐4 inhibition rate included area under the effect–time curve over168 h (AUEC_0–168 h), maximum inhibition rate (EC_max), minimum inhibition rate (EC_min), and the time to maximum inhibition rate (ET_max).

Blood samples (2 mL) were collected at the fasting state and at 10 min, 20 min, 0.5 h, 1 h, 1.5 h, 2 h, 3 h, and 4 h after glucose solution ingestion on Day 1 to determine plasma glucose concentrations, insulin, and C‐peptide levels. The oral glucose tolerance test (OGTT) was performed on Days 4, 27, and 41, and plasma glucose concentrations were measured at the fasting state and at 30, 60, 120, and 240 min after glucose solution ingestion. The PD parameters included area under the effect–time curve (AUEC) over the dosing interval, maximum glucose concentration (EC_max), and time to EC_max (T_max).

Safety Measurements

Safety was assessed through the monitoring of adverse events (AEs), serious adverse events (SAEs), and treatment‐emergent adverse events (TEAEs). All AEs are assessed in terms of their severity, intensity (mild, moderate, or severe), and relationship to the study drug. The encoding of AE was defined by the conditions in accordance with the Common Terminology Criteria for the Medical Dictionary for Regulatory Activities (MedDRA) v. 22.1. The physical examination, laboratory examination, vital signs, 12‐ECG, and hypoglycemic events were also performed at each visit.

Sample Size

The sample size was determined in accordance with the “Guidelines for Phase I Clinical Trial Management of Drugs (Trial)” (2011) issued by China's National Medical Products Administration (NMPA). It was estimated that enrolling in 17 subjects would be able to detect a difference of at least 5% in the PK parameters of interest with 85% power, considering an intrasubject coefficient of variation (CV%) of 20%. Considering a dropout rate of 20%, a total of 22 subjects were enrolled in the study.

Statistical Analysis

Statistical analyses were performed using SAS software (version 9.4; SAS Institute, Inc., Cary, NC). PK and PD analysis was performed on data from the subjects who received at least one dose of study drug. Demographic data, PK and PD, and safety assessments were assessed in a descriptive manner. Continuous variables were described as mean ± standard deviation (SD). Qualitative variables were presented as number (%). PK and PD parameters were calculated by Phoenix WinNonlin (version 8.1, Pharsight Corporation, Mountain View, CA). No significant DDI was concluded if the 90% CI for the geometric mean ratios (GMR) of the PK and PD parameters for the combination therapy versus cofrogliptin or metformin monotherapy were within 0.8–1.25 fold.

Results

Subject Demographics

A total of 55 healthy Chinese adult subjects were screened, among which 33 subjects did not meet eligibility criteria. Consequently, 22 subjects (11 male and 11 female) were enrolled in the study. The mean age of the subjects was 33.9 ± 7.05 years (range 23–48 years), mean BMI was 24.2 ± 1.89 kg/m² (range 20.5–27.6 kg/m²). Among the enrolled subjects, one subject had a history of appendectomy and four subjects reported the concomitant medication use. The baseline demographic and clinical characteristics of all enrolled subjects are summarized in Table 1. One subject voluntarily withdrew from the study during treatment period 1, 21 subjects completed the study and were included in the PK and PD assessments.

Table 1.

Baseline Demographic and Clinical Characteristics of All Enrolled Subjects

Variable (n = 22)	Value
Age, years, mean ± SD (range)	33.9 ± 7.05 (23–48)
Height, cm, mean ± SD (range)	164 ± 0.06 (153–173)
Weight, kg, mean ± SD (range)	64.9 ± 7.16 (52–79.7)
BMI, kg/m2, mean ± SD (range)	24.2 ± 1.89 (20.5–27.6)
Sex, n (%)
Male	11 (50)
Female	11 (50)
Fertility status, n (%)
Yes	12 (54.6)
No	10 (45.5)
Ethnicity, n (%)
Chinese‐Han	22 (100)
Others	0
Medical history (%)
Yes	1 (4.5)
No	21 (95.5)
Concomitant medication (%)
Yes *	4 (18.2%)
No	18 (81.8)

BMI, body mass index; SD, standard deviation.

^*Concomitant medication: two subjects, Compound lidocaine cream; one subject, Penciclovir cream and Chonglou Jiedu Ding; one subject, 50% glucose injection.

PK Assessment

Effect of Cofrogliptin on Metformin PK Properties

The mean plasma concentration–time profiles (linear and semilogarithmic) for metformin monotherapy (Day 4) and combination therapy (Day 41) are shown in Figure 2A,B. On Day 4, plasma concentrations of metformin reached a steady state, with C_{ss (max)} value of 2480 ± 705 ng/mL. When metformin was administered with cofrogliptin, metformin C_{ss (max)} value reached 2112 ± 797 ng/mL. Mean metformin exposure (AUC_0–8 h) on Day 4 and Day 41 was 10,630 ± 2884 and 9127 ± 1835 h ng/mL, respectively. Metformin Ae_0–8 h and Ratio_0–8 h values on Day 4 and Day 41 were 138,782 ± 49,269 g and 0.426 ± 0.144 % and 125,743 ± 48,568 g and 0.431 ± 0.208 %, respectively. The mean cumulative urinary excretion rate–time curves of metformin on Day 4 and Day 41 are shown in Figure 3A.

Figure 2.

(A for liner scale, B for semi‐log) Mean metformin plasma concentration–time profiles after metformin monotherapy (Day 4) or metformin plus cofrogliptin (Day 41). (C for liner scale, D for semi‐log) Mean cofrogliptin plasma concentration–time profiles after cofrogliptin monotherapy (Day 27) or metformin plus cofrogliptin (Day 41). The grey dashed line represents the overall mean trend of all curves in each panel.

Figure 3.

(A) The mean cumulative urinary excretion rate–time curves of metformin on Day 4 and Day 41. (B) The glucose concentration–time profile during the OGTT for metformin monotherapy and metformin plus cofrogliptin (Day 1, Day 4, and Day 41). (C) The glucose concentration–time profile during the OGTT for cofrogliptin monotherapy and cofrogliptin plus metformin (Day 1, Day 27, and Day 41). (D) The DPP‐4 inhibition rate–time curve graph for cofrogliptin monotherapy (Day 27) and cofrogliptin plus metformin (Day 41). The grey dashed line represents the overall mean trend of all curves in each panel.

The detail PK parameters along with the results of statistical analysis are summarized in Table 2. The 90% CIs of the GMRs of C_{SS (max)} and Ae_0–8 h were 0.840 (0.743, 0.950) and 0.899 (0.782, 1.032), respectively, slightly extending the lower limit of 90% CI for the predefined DDI criteria for no interaction. However, the GMRs (90% CI) of the combination therapy over metformin monotherapy were 0.872 (0.809, 0.941), 0.900 (0.834, 0.971), and 0.994 (0.871, 1.136) for AUC_0–8 h, AUC_0–12 h, Ae Ratio_0–8 h, respectively, falling within 0.80–1.25 fold. These results indicated that cofrogliptin could slightly reduce the peak plasma concentrations of metformin, but it did not significantly affect the systemic exposure or renal excretion of metformin.

Table 2.

Pharmacokinetic Parameters in Metformin or Cofrogliptin Monotherapy and Combination Therapy

	Metformin PK Parameters			Cofrogliptin PK Parameters
Parameter (Unit)	Metformin	Metformin + Cofrogliptin	GMR (90% CI)	Cofrogliptin	Cofrogliptin + Metformin	GMR (90% CI)
AUC0–8 h (h ng/mL)	10630 ± 2884	9127 ± 1835	0.872 (0.809, 0.941)	‐	‐	‐
AUC0–12 h (h ng/mL)	11999 ± 3301	10648 ± 2250	0.900 (0.834, 0.971)	‐	‐	‐
AUCtau (h ng/mL)	‐	‐	‐	48182 ± 11870	54197 ± 14779	1.12 (1.072, 1.169)
Css (max) (ng/mL)	2480 ± 705	2112 ± 797	0.840 (0.743, 0.950)	533 ± 118	600 ± 97.3	1.14 (1.057, 1.220)
Ae0–8 h (µg)	138782 ± 49269	125743 ± 48568	0.899 (0.782, 1.032)	‐	‐	‐
Ratio0–8 h (%)	0.426 ± 0.144	0.431 ± 0.208	0.994 (0.871, 1.136)	‐	‐	‐
AUC%Extap (%)	8.59 ± 2.57	22.9 ± 11.1	‐	28.5± 7.29	29.2 ± 8.95	‐
AUCinf (h ng/mL)	13151 ± 2725	12223 ± 3539	‐	69381 ± 22871	79949 ± 31606	‐
CL/F (L/h)	73.3 ± 20.7	81.1 ± 17.9	‐	0.553 ± 0.151	0.496 ± 0.141	‐
Css (avr) (ng/mL)	1143 ± 313	1004 ± 206	‐	287 ± 70.7	323 ± 88	‐
Cmax (ng/mL)	2480 ± 705	2112 ± 797	‐	533 ± 118	600 ± 97.3	‐
Css (min) (ng/mL)	193 ± 57.8	240 ± 108	‐	107 ± 36	118 ± 50.5	‐
Kel (1/h)	0.218 ± 0.033	0.222 ± 0.057	‐	0.008 ± 0.002	0.008 ± 0.002	‐
Tmax (h)	1.0 (0.5–4.0)	1.0 (0.5–5.0)	‐	4.0 (2.0–24.0)	4.0 (4.0–24.0)	‐
Vz/F (L)	343 ± 110	413 ± 264	‐	69.9 ± 11.7	64.7 ± 13	‐
T1/2 (h)	3.25 ± 0.471	3.58 ± 2.19	‐	91.1 ± 18.7	94.9 ± 24.2	‐
CLR (L/h)	14.0 ± 5.15	14.2 ± 6	‐	‐	‐	‐
Ae0–12 h (µg) *	157275 ± 47879	‐	‐	‐	‐	‐
Ratio0–12 h (%)	0.313 ± 0.114	‐	‐	‐	‐	‐

Ae_0–8 h or Ae_0–12 h,, cumulative urinary excretion Ae in 8 or 12 h; AUC_0–8 h, area under the plasma concentration–time curve over 8 h; AUC_0–12 h, area under the plasma concentration–time curve over 12 h; AUC_tau, area under the plasma concentration versus time curve during the dosing interval at steady state; AUC_%Extap, extrapolated area under the plasma concentration–time curve; AUC_inf, area under the plasma concentration–time curve from zero to infinity; CI, confidence interval; CL/F, apparent clearance; C_max, maximum plasma concentration; C_{ss (avr)}, average plasma concentration at steady state; C_{ss (max)} maximum steady‐state concentration; C_{ss (min)}, minimum steady‐state concentration; GMR, geometric mean ratios; Kel, elimination rate constant; T_max, time to C_max; T_1/2, apparent terminal elimination half‐life; Vz/F, apparent volume of distribution.

^*Owing to repeated administration of metformin over a long period and the 10‑h dosing interval in the study, samples collected 12 h after the last dose will be affected by the second dose. Therefore, Ae₀_–₁_{2 h} is not calculated in the metformin pharmacokinetic (PK) parameters on Day 41 (metformin + cofrogliptin).

Data are mean ± SD for all except for T_max, which is median (range).

Effect of Metformin on Cofrogliptin PK Properties

The mean plasma concentration–time profiles (linear and semilogarithmic) for cofrogliptin monotherapy (Day 27) and combination therapy (Day 41) are shown in Figure 2C,D. The PK parameters are summarized in Table 2. On Day 27, plasma concentrations of cofrogliptin reached a steady state, with C_{ss (max)} value of 533 ± 118 ng/mL. When cofrogliptin was administered with metformin, C_{ss (max)} value of cofrogliptin was 600 ± 97.3 ng/mL. Mean cofrogliptin exposure on Day 27 and Day 41 was 48,182 ± 11,870 and 54,197 ± 14,779 h ng/mL, respectively. Notably, the GMR (90% CI) for AUC_tau and C_{ss (max)} for cofrogliptin plus metformin/cofrogliptin monotherapy were 1.12 (1.072, 1.169) and 1.14 (1.057, 1.220), respectively, both were entirely within 0.80–1.25 fold. This indicated that metformin did not significantly affect the PK properties of cofrogliptin.

PD Assessment

Effect of Cofrogliptin on Metformin PD Properties

Following oral glucose administration on Day 1, the EC_max, AUEC_0–4 h and AUEC_0–0.5 h of insulin and C‐peptide were 109 ± 56.5 mU/L and 2723 ± 776 mU/L, 189 ± 80.1 h mU/L, and 6462 ± 1695 h pmol/L, 25.4 ± 10.9 h mU/L, and 594 ± 158 h pmol/L, respectively. The glucose concentration–time profiles during the OGTT are shown in Figure 3B,C. The PD parameters of plasma glucose are shown in Table 3. When metformin was administered alone on Day 4, the changes from baseline of plasma glucose AUEC_0–4 h, AUEC_0–0.5 h, and EC_max were 1.76 ± 3.33 h mmol/L, −0.129 ± 0.402 h mmol/L, and 0.595 ± 1.29 mmol/L, respectively. In comparison with the corresponding values of cofrogliptin monotherapy on Day 27, the changes of glucose AUEC_0–4 h, AUEC_0–0.5 h, and EC_max were −0.219 ± 2.54 h mmol/L, −0.144 ± 0.311 h mmol/L, and −0.080 ± 1.67 mmol/L, respectively, when cofrogliptin was administered with metformin (Day 41).

Table 3.

The Pharmacodynamic Parameters of Glucose and DPP‐4 Inhibition Rate

Parameter (unit)	Baseline	Metformin	Cofrogliptin	Cofrogliptin + Metformin	GMR a (90% CI)	GMR b (90% CI)	GMR c (90% CI)
Glucose
AUEC0‐0.5 h (hr×mmol/L)	3.68 ± 0.261	3.41 ± 0.372 α	3.21 ± 0.339	3.07 ± 0.258 β	0.955 (0.914, 0.998)	0.915 (0.879, 0.952)	‐
AUEC0‐4 h (hr×mmol/L)	25.9 ± 3.03	27.7 ± 3.24	24.1 ± 2.73	23.9 ± 2.24 β	1.06 (1.009, 1.111),	0.870 (0.833, 0.908)	‐
ECmax (mmol/L)	9.34 ± 1.16	9.97 ± 1.10	8.62 ± 1.59	8.54 ± 1.17 β	1.07 (1.010, 1.128)	0.866 (0.815, 0.920)	‐
Tmax (hr)	0.5 (0.33–1)	1 (0.5–2)	1 (0.5–4)	1 (0.5–2)	‐	‐	‐
ΔAUEC0‐4 h (hr×mmol/L)	‐	1.76 ± 3.33 #	‐	−0.219 ± 2.54 *	‐	‐	‐
ΔAUEC0‐0.5 h (hr×mmol/L)	‐	−0.129 ± 0.402 #	‐	−0.144 ± 0.311 *	‐	‐	‐
ΔECmax(mmol/L)	‐	0.595 ± 1.29 #	‐	−0.080 ± 1.67 *	‐	‐	‐
DPP‐4 inhibition rate
AUEC0‐168 h (hr×%)	‐	‐	15800 ± 641	15944 ± 543	‐	‐	1.01 (1.003, 1.015)
ECmax (%)	‐	‐	97.1 ± 2.19	97.6 ± 2.05%	‐	‐	1.00 (1.001, 1.009)
ECmin (%)	‐	‐	87 ± 7.42	89.5 ± 6.54	‐	‐	1.03 (1.005, 1.040)
ETmax (hr)	‐	‐	24 (2–72)	24 (2–72)	‐	‐	‐

AUEC, area under the effect–time curve over the dosing interval (0‐0.5, 0‐4, and 0‐168 h); CI, confidence interval; GMR, geometric mean ratios; EC_max, maximum effect; T_max, time to EC_max.

^#AUEC and EC_max change from baseline.

^*The difference values of AUEC and EC_max between combination therapy and cofrogliptin monotherapy.

^aGMR of metformin to baseline.

^bGMR of Cofrogliptin + Metformin to Metformin.

^cGMR of Cofrogliptin + Metformin to Cofrogliptin. Data are mean ± SD for all except for T_max and ET_max, which are median (range).

^α P <  .05, Metformin versus Baseline.

^β P <  .05, Metformin versus Cofrogliptin + Metformin.

The plasma glucose during the OGTT of metformin monotherapy (Day 4) did not show significant differences from baseline, as the 90% CIs of the AUEC_0–4 h, AUEC_0–0.5 h, and EC_max of the metformin to baseline—(1.009, 1.111), (0.914, 0.998), and (1.010, 1.128), respectively—remained within 0.80–1.25 fold. These results indicated that the hypoglycemic effect of metformin was not obvious in healthy subjects and only showed a certain hypoglycemic effect within 0.5 h after administration (AUEC_0–0.5 h, 3.41 ± 0.372 h mmol vs 3.68 ± 0.261 h mmol, P <  .01). In comparison with metformin monotherapy, the hypoglycemic effect was further enhanced after the combination therapy (AUEC_0–0.5 h, 3.07 ± 0.258 vus 3.41 ± 0.372 h mmol; AUEC_0–4 h, 23.9 ± 2.24 h mmol vs 27.7 ± 3.24 h mmol; EC_max 8.54 ± 1.17 mmol/L vs 9.34 ± 1.16 mmol/L; all P <  .01). While the 90% CIs of the AUEC_0–0.5 h, AUEC_0–4 h, and EC_max of the combination therapy versus metformin monotherapy—0.879, 0.952), (0.833, 0.908), and (0.815, 0.920), respectively—were all within 0.80–1.25 fold (Table 3). Overall, cofrogliptin did not result in clinically significant differences in the hypoglycemic efficacy of metformin.

Effect of Metformin on Cofrogliptin PD Properties

The DPP‐4 inhibition rate–time curve for cofrogliptin monotherapy and combination therapy are shown in Figure 3D. When administered alone or in combination with metformin, cofrogliptin achieved DPP‐4 inhibition rate EC_max values of 97.1 ± 2.19% and 97.6 ± 2.05%, respectively, and AUEC_0–168 h values of 15,800 ± 641 hr×% and 15,944 ± 543 hr×%, respectively. The GMRs (90% CI) of combination therapy over cofrogliptin monotherapy in terms of DPP‐4 inhibition rate were 1.01 (1.003, 1.015), 1.00 (1.001, 1.009), and 1.03 (1.005, 1.040) for AUEC_0–168 h, EC_max, and EC_min, respectively, thus, meeting the DDI criteria (0.8–1.25) for no significant interaction (Table 3). This indicated that metformin did not significantly affect the DPP‐4 inhibition rate of cofrogliptin.

Safety

All 22 subjects enrolled were included in the safety analysis. The safety results are summarized in Table 4. A total of 385 AEs (including 378 TEAEs) were reported in all subjects (n = 22). Of the reported TEAEs, 374 (98.94%) were Grade 1 in severity (mild), while 4 (1.06%) were classified as Grade 2 (moderate). Regarding AE of special interest, hypoglycemia was reported in 10 subjects (45.45%). However, none of these events were severe. During cofrogliptin monotherapy, the most frequent TEAEs (≥10%) were metabolic and nutritional disorders, with hypoglycemia occurring in 23.81% of subjects. In contrast, during the combination therapy, the most frequent TEAEs (≥10%) included gastrointestinal disorders (nausea, 80.95%; diarrhea, 80.95%; abdominal pain, 57.14%; abdominal distension, 23.81%; vomiting, 28.57%; and gastroesophageal reflux disease, 19.05%), metabolism and nutrition disorders (hypoglycemia, 23.81% and anorexia, 61.90%), systemic diseases and various reactions at the administration site (asthenia, 23.81%), and laboratory abnormalities (23.81%). All AEs of less than moderate severity were resolved without sequelae. No deaths, SAEs, or TEAEs leading to discontinuation were reported. No clinically meaningful changes were observed in the results of vital signs, physical examination, laboratory tests, and 12‐ECG during the study. Overall, cofrogliptin and metformin administered alone or in combination were safe and well tolerated in healthy Chinese subjects.

Table 4.

Summary of adverse Events During Any of the Study Periods

Variables, n (%)	Metformin (n = 22)	Cofrogliptin (n = 21)	Metformin + Cofrogliptin (n = 21)
Any TEAE	18 (81.8)	13 (61.9)	21 (100)
AE of special interest
Hypoglycemia	4 (18.2)	5 (23.81)	6 (28.57)
Severe hypoglycemia	0 (0)	0 (0)	0 (0)
TEAEs in ≥5% of subjects *
Gastrointestinal system diseases	18 (81.82)	1 (4.76)	20 (95.24)
Diarrhea	17 (77.27)	0 (0)	17 (80.95)
Nausea	10 (45.45)	0 (0)	17 (80.95)
Abdominal pain	7 (31.82)	0 (0)	12 (57.14)
Vomiting	3 (13.64)	0 (0)	6 (28.57)
Abdominal distension	0 (0)	0 (0)	5 (23.81)
Gastroesophageal reflux disease	1 (4.55)	0 (0)	4 (19.05)
Abdominal pain upper	0 (0)	0 (0)	2 (9.52)
Indigestion	0 (0)	0 (0)	2 (9.52)
Metabolic and nutritional diseases	4 (18.18)	5 (23.81)	16 (76.19)
Anorexia	0 (0)	0 (0)	13 (61.90)
Hypoglycemia	4 (18.18)	5 (23.81)	6 (28.57)
Nervous system disease	2 (9.09)	2 (9.52)	3 (14.29)
Headache	0 (0)	1 (4.76)	2 (9.52)
Systemic diseases and various reactions at the administration site	0 (0)	2 (9.52)	6 (28.57)
Asthenia	0 (0)	1 (4.76)	5 (23.81)
Heart disease	1 (4.55)	2 (9.52)	2 (9.52)
Injury, poisoning, and procedural complications	0 (0)	1 (4.76)	2 (9.52)
Ophthalmic complications	0 (0)	0 (0)	2 (9.52)
Skin and subcutaneous diseases	2 (9.09)	1 (4.76)	2 (9.52)
Rash	2 (9.09)	2 (9.52)	0 (0)
Abnormal inspections	2 (9.09)	6 (28.57)	16 (76.19)

AE, adverse event; SAE, serious AE; TEAE treatment‐emergent adverse event.

*AEs classified by System Organ Classes and Preferred Term.

Discussion

DPP‐4 inhibitors regulate glucose levels by enhancing incretin activity, while metformin reduces hepatic glucose output and improves insulin sensitivity. ²⁹ Their independent actions and different excretion pathways reduce the possibility of DDI. Clinical studies have confirmed that the combination of DPP‐4 inhibitors and metformin could provide effective glycemic control with a favorable safety profile and low drug interaction risk. ²⁵ , ²⁶ , ²⁹ , ³⁰ , ³¹ Cofrogliptin, a novel DPP‐4 inhibitor, has demonstrated promising efficacy and safety in preliminary trials. ²⁰ , ²² , ²⁴ These findings highlight the need to investigate its potential DDI when combined with metformin. In this study, we evaluated whether the combination of cofrogliptin and metformin influenced the PK, PD, or safety profile of each other. These findings could potentially support the combination therapy as a potential treatment regimen for T2DM.

Our study was carefully designed to thoroughly investigate the potential DDI between cofrogliptin and metformin. An initial dose of 35 mg of cofrogliptin followed by 25 mg (once weekly) and metformin 1000 mg BID were chosen to maximize the potential drug interactions. Metformin is transported into hepatocytes by organic cation transporter 1 (OCT1) and out of the hepatocytes by multidrug and toxin extrusion 1 (MATE1), which facilitates its hypoglycemic therapeutic effects. ³² , ³³ Consequently, the plasma concentration of metformin does not fully represent its impact on renal active transporters. To assess the effect of cofrogliptin on the active renal secretion of metformin, urinary PK data were collected following metformin administration. Additionally, OGTT was conducted to evaluate the effect of cofrogliptin on the hypoglycemic effects of metformin. The hypoglycemic effect of cofrogliptin is assessed by DPP‐4 inhibition rate. Consequently, the PD parameters related to DPP‐4 inhibition rate after the administration of cofrogliptin were utilized to evaluate the PD effects of metformin on cofrogliptin.

In this study, the co‐administration of cofrogliptin and metformin led to a slight decline in maximum steady‐state concentration (C_{ss (max)}) of metformin, which is consistent with findings from studies investigating the combination therapy of gemigliptin and metformin. ³⁰ This mild reduction in plasma metformin concentrations at 1–4 h post‐dose is a common clinically insignificant PK pattern reported for other DPP‐4 inhibitors (e.g., evogliptin and teneligliptin) in combination with metformin. ²⁸ , ³⁴ Although the 90% CI for the GMR of metformin C_{ss (max)} did not fell within the 0.80–1.25 range, the fact that metformin AUC_0–8 h and AUC_0–12 h showed no significant alteration in combination with cofrogliptin suggests that this slight decline in metformin C_{ss (max)} is unlikely to be clinically significant. Meanwhile, the amount of metformin excreted by urine from 0 to 8 h was comparable between metformin monotherapy and the combination therapy, with 90% CI for the GMR falling within the 0.8–1.25 range. The plasma concentration profile of cofrogliptin was similar between cofrogliptin monotherapy and co‐therapy of gemigliptin and metformin, and the 90% CIs for the GMR of cofrogliptin AUC_tau and C_{ss (max)} were within the 0.80–1.25 range. The results indicated that cofrogliptin and metformin did not exhibit clinically significant PK interactions. These outcomes are consistent with data from DDI studies of other DPP‐4 inhibitors, including gemigliptin, fotagliptin, and teneligliptin, which analogously showed no PK interactions in combination therapy with metformin. ³⁰ , ³¹ , ³⁴

PK drug interactions are only clinically meaningful when the glucose regulation (PD) was also altered. ³⁵ Thus, in this study, OGTT was performed to explore the effect of cofrogliptin on glucose regulation of metformin. The AUC of glucose during the first 30 min after glucose intake was significantly reduced after the administration of metformin (3.41 ± 0.372 vs 3.68 ± 0.261, P <  .01), which was consistent with the previous study. ³⁶ The co‐administration of metformin and cofrogliptin resulted in a further reduction in the AUC for glucose. However, the 90% CIs for glucose AUEC_0–4 h, AUEC_0–0.5 h, and EC_max for combination therapy to metformin monotherapy all fell within the 0.80–1.25 range. These results indicated that cofrogliptin did not cause clinically significant changes in the hypoglycemic effect of metformin. Cofrogliptin exerts its hypoglycemic effect mainly by inhibiting DPP‐4 activity. ²⁰ In this study, the inhibition of DPP‐4 activity by cofrogliptin was comparable whether administered alone or in combination with metformin, suggesting meaningful PD interaction between the two agents. Furthermore, the DPP‐4 inhibition rate (>90%) indicates a meaningful pharmacodynamic interaction between the two agents. ³⁷ In summary, the early reduction in plasma metformin concentration at 2–4 h during co‐administration did not translate into altered systemic exposure (AUC), and the complementary improvements in glucose control are attributable to the independent but complementary PD mechanisms of cofrogliptin and metformin, rather than a mutual interaction between the two drugs.

Hypoglycemia was an expected AE of all hypoglycemic agents. The higher incidence of mild hypoglycemia may be caused by the sympatho‐inhibitory effect of higher doses of metformin, which might increase the risk of hypoglycemia in healthy subjects. ³⁸ Gastrointestinal disorders (such as nausea, abdominal pain, abdominal distension, and vomiting) were the most frequently reported AEs. In this study, metformin was initiated at a dose approximating the clinical maximum tolerated level, without employing a titration period. Consequently, this approach led to a notable increase in gastrointestinal adverse reactions over time. ³⁹ , ⁴⁰ No subject experienced gastrointestinal disorders during cofrogliptin monotherapy, which was consistent with the results in Phase I and II clinical studies of cofrogliptin (unpublished data). Although the incidence of gastrointestinal disorders, anorexia, and asthenia was numerically higher in the combination phase, these events were all mild or moderate in severity. The increased incidence of AEs during combination therapy may be related to interindividual variability or additive PD effects of the two agents, rather than a specific PK or PD DDI. When cofrogliptin is administered with high‑dose metformin, we recommend monitoring for any changes in AEs, particularly gastrointestinal disorders.

This study has several limitations. The trial was conducted in healthy subjects, the extrapolation of drug effects to patients with T2DM is limited. There are inherent physiological and baseline differences between healthy subjects and T2DM patients (e.g., cardiovascular/vascular complications, altered baseline glucose levels, and organ impairment), which may potentially lead to different clinical outcomes in the clinical setting. Therefore, further studies to investigate the clinical efficacy of the combination of cofrogliptin and metformin in T2DM patients are needed.

Conclusions

In summary, the combination of cofrogliptin and metformin did not result in any clinically significant PK or PD interactions in healthy Chinese subjects. Cofrogliptin monotherapy or combination therapy with metformin was safe and well tolerated in healthy Chinese subjects.

Author Contributions

Cheng Cui and Na Liu wrote the manuscript. Dongyang Liu and Haiyan Li designed the research. Cheng Cui, Na Liu, Yang Huang, and Shu Niu performed the research. Cheng Cui, Na Liu, Shu Niu, Fangqiong Li, Yaming Li, Dongyang Liu, and Haiyan Li analyzed the data. Chang Chu revised the manuscript. All authors have given final approval of the version to be published.

Conflicts of Interest

Chang Chu, Fangqiong Li and Yaming Li were employees of the Haisco Pharmaceutical Group Co., Ltd. Their involvement did not influence the integrity and objectivity of the research findings presented in this study. The remaining authors declare no competing financial interests.

Funding

This study was sponsored by Haisco Pharmaceutical Group Co. Ltd ., the manufacturer of cofrogliptin.

Data Availability Statement

The data and materials used and/or analyzed during the present study are available from the corresponding author on reasonable request.