Dorzagliatin, a Dual-Acting Glucokinase Activator, Increases Insulin Secretion and Glucose Sensitivity in Glucokinase Maturity-Onset Diabetes of the Young and Recent-Onset Type 2 Diabetes

Individuals with maturity-onset diabetes of the young due to GCK inactivating mutations (GCK-MODY) have a marked reduction in β-cell glucose sensitivity (βCGS) and impaired α-cell glucose sensing (1). Until now, there are no effective glucose-lowering drugs to correct the primary defect of impaired glucose sensing in GCK-MODY. Dorzagliatin is a novel, first-in-class, dual-acting allosteric glucokinase (GK) activator (GKA). It directly binds to a pocket distal to the active site of GK, increases its affinity for glucose, and lowers the set point for glucose-stimulated insulin secretion. In a phase 3, double-blind, placebo-controlled trial among treatment-naive patients with type 2 diabetes, dorzagliatin (75 mg twice daily) lowered HbA_1c by −1.07% (−11.77 mmol/mol) vs. −0.50% (−5.5 mmol/mol) in the placebo group (P < 0.0001) (2). Dorzagliatin also improved HbA_1c compared with placebo in patients with type 2 diabetes on metformin therapy, with efficacy maintained up to 52 weeks (3). Based on oral glucose tolerance test (OGTT)–derived indices, treatment with dorzagliatin 75 mg twice daily for 28 days improved HOMA of β-cell function and early insulin secretion (insulinogenic index) (4). However, β-cell glucose sensing and β-cell secretory capacity are separate defects in type 2 diabetes (5) that cannot be easily dissected using OGTT alone.

In this double-blind, placebo-controlled, crossover study, we evaluated the efficacy of dorzagliatin in patients with GCK-MODY and in patients with recent-onset type 2 diabetes using the hyperglycemic glucose clamp technique. We hypothesized that dorzagliatin would improve βCGS and insulin secretion in both groups to different degrees. Individuals who are heterozygous for GCK mutations might have increased responsiveness to dorzagliatin through upregulation of wild-type GK activity, analogous to hypersensitivity to sulfonylureas in patients with transcription factor MODY (6). Alternatively, dorzagliatin may be able to restore mutant GK activity through direct action on the allosteric site (7). To test this hypothesis, we conducted in vitro studies on enzyme kinetics of wild-type and mutant GK carried by patients with GCK-MODY who underwent the clamp study. This novel information may contribute to development of precision medicine in diabetes.

This was a randomized, double-blind, crossover study in 8 patients with GCK-MODY and 10 patients with type 2 diabetes. All participants received a single oral dose of dorzagliatin 75 mg or matched placebo followed by a 2-h hyperglycemic clamp on two occasions separated by a 2-week washout period. The study design is summarized in Supplementary Fig. 1. The study was approved by the Joint New Territories East Cluster–The Chinese University of Hong Kong Clinical Research Ethics Committee (CREC 2020.196). All participants provided written informed consent. The study was conducted at the Phase 1 Clinical Trial Centre, The Chinese University of Hong Kong. Participants were recruited between 30 September 2020 and 23 August 2021, and clinical data were collected between 12 October 2020 and 23 September 2021.

All participants had physician-diagnosed type 2 diabetes or GCK-MODY and were attending the Prince of Wales Hospital, Shatin, in Hong Kong or were family members of GCK-MODY probands. All participants were aged ≥18 and <65 years and had BMI of >18 and <30 kg/m². Exclusion criteria included body weight ≥45 kg, pregnancy or lactating, stroke or cardiovascular disease within 6 months of recruitment, severe renal dysfunction (estimated glomerular filtration rate <30 mL/min/1.73 m² or renal replacement therapy), severe hepatic dysfunction (AST and/or ALT greater than three times the upper limit of normal), history of drug abuse or excessive alcohol intake, severe hypoglycemia within 3 months before screening, anemia, excessive blood loss >300 mL, and use of strong or moderate CYP3A4 inhibitors or inducers. Use of sulfonylureas, dipeptidyl peptidase 4 inhibitors, glucagon-like peptide 1 agonists, sodium–glucose cotransporter 2 inhibitors, insulin, thiazolidinediones, or acarbose in the 3 months before study enrollment were not permitted. Participants with type 2 diabetes had the diagnosis for at least 3 months with <2 years with HbA_1c ≥6.5% (48 mmol/mol) and <8% (64 mmol/mol) for diet control and ≥6.0% (42 mmol/mol) and <8% (64 mmol/mol) for treatment with metformin. Participants with GCK-MODY had a fasting plasma glucose >5.6 mmol/L and were heterozygous carriers of a pathogenic or likely pathogenic GCK mutation at screening based on guidelines published by the American College of Medical Genetics and Genomics (8), Association for Clinical Genomic Science (9), and the ClinGen Monogenic Diabetes Expert Panel (10).

During the screening visit (visit 1), demographic data, medical and family history, and physical examination were performed to determine eligibility. Fasting plasma glucose, HbA_1c, complete blood count, renal and liver function tests, lipid profile, and urinary pregnancy test in women with reproductive potential were performed for assessment of eligibility. A blood sample was collected for DNA extraction for genotyping of monogenic diabetes.

The study drugs were dorzagliatin 75-mg tablet (HMS5552; Shanghai Desano Bio-Pharmaceutical Co., Ltd.) and matched placebo (Shanghai Desano Bio-Pharmaceutical Co., Ltd.). Each eligible participant was randomly assigned to receive either dorzagliatin 75 mg or matching placebo on two occasions, separated by 14 ± 2 days. The treatment sequence was pregenerated randomly by a computer program by personnel independent of the study team. Each participant received the study drug from pack 1 on the first clamp visit (visit 2) and from pack 2 on the second clamp visit (visit 3). All study investigators, study personnel, monitors, and participants were masked to the treatment sequence except for unblinding due to emergency management.

Before each hyperglycemic clamp visit (visits 2 and 3), participants were fasted for at least 8 h overnight and asked to refrain from smoking, alcohol, and vigorous exercise for 48 h before dosing. Metformin, if taken, was withheld for 72 h before dosing. In healthy volunteers, the time to maximum (T_max) of dorzagliatin was 90 min (11). All participants received a single oral dose of dorzagliatin 75-mg tablet or placebo with 240 mL of water 90 min before (−90 min) commencement of the hyperglycemic clamp (0 min). Blood samples for glucose, insulin, C-peptide, and glucagon were collected from a retrograde cannula inserted into the dominant hand/wrist vein placed in an insulated warm box with air heated to 40–50°C for arterialization of venous blood. Capillary blood glucose was monitored between −90 and −20 min, targeting a blood glucose of 5–6 mmol/L at 0 min. A bolus of 20% dextrose was administered intravenously if consecutive blood glucose values were <3.5 mmol/L. A dose of intravenous regular insulin was administered for elevated basal blood glucose levels (>6.0 mmol/L), which was stopped at least 20 min before clamp start.

Blood samples were collected at −20, −10, and 0 min to define baseline plasma glucose, insulin, and C-peptide levels. At time 0, a primed dose of 20% dextrose was given over 15 min through an infusion pump to rapidly raise the arterialized whole-blood glucose to a target of 12 mmol/L. Blood glucose was thereafter maintained at 12 mmol/L for 120 min by titration of dextrose infusion rates according to arterialized blood glucose level measured in duplicate every 5 min using a bedside glucose analyzer (YSI 2300; Yellow Springs Instruments) (coefficient of variation [CV] <2%). Blood samples were obtained for insulin and C-peptide every 2 min in the first 10 min and thereafter at 20, 40, 80, 100, and 120 min to assess second-phase insulin response. Glucagon was measured at 0, 10, 20, 40, 80, and 120 min. At the end of the clamp, 20% dextrose infusion was gradually switched off over 30 min, followed by monitoring of blood glucose for at least 30 min until return to euglycemic levels. After a washout period of 14 ± 2 days, participants underwent a second hyperglycemic clamp (visit 3) with either dorzagliatin or placebo administration, depending on sequence randomization. After each clamp study, all participants self-monitored blood glucose twice daily for 48 h for reporting of any hypoglycemic event, with phone follow-up (visit 4) 3–7 days after last dosing to ascertain any adverse events.

Blood samples for hormonal assays were collected in chilled EDTA tubes and then centrifuged at 3,000 rpm for 10 min at 4°C. C-peptide was measured spectrophotometrically using C-peptide ELISA kits (cat. no. 10-1136-01; Mercodia AB, Uppsala, Sweden) (intra-assay CV 3.2%, interassay CV 4.8%). Insulin was measured spectrophotometrically using insulin ELISA kits (cat. no. 10-1113-01; Mercodia AB) (intra-assay CV 3.3%, interassay CV 6.1%). Glucagon was measured spectrophotometrically using glucagon ELISA kits (cat. no. 10-1271-01; Mercodia AB) (intra-assay CV 8.3%, interassay CV 9.2%). Absorbance at 450 nm was read on a Multiskan FC Microplate Photometer (Thermo Fisher Scientific, Waltham, MA).

At the screening visit, a 6-mL peripheral blood sample was collected from each participant, and DNA was extracted using MagNA Pure 96 System (Roche Diagnostics International, Rotkreuz ZG, Switzerland). GCK variants were identified by next-generation sequencing. Additionally, we included other genes related to monogenic diabetes using a custom-targeted DNA panel covering 34 known genes for monogenic diabetes (Supplementary Methods). The sequencing regions covered exons and flanking regions located within 25 base pairs upstream and downstream of each exon. DNA libraries were prepared according to the reference guide of AmpliSeq for Illumina Custom and Community Panels. Pooled libraries were sequenced using a MiSeq instrument (Illumina, San Diego, CA) with paired-end run of 151 cycles, with FASTQ files as raw sequencing output per read. Sequencing variants that passed quality control were interpreted according to published guidelines (8–10).

Basal glucose was determined as mean glucose levels (area under the curve [AUC]/time) from −20 to 0 min. Steady-state glucose during the hyperglycemic clamp was determined as mean glucose levels (AUC/time) from 80 to 120 min. AUC was calculated using the trapezoidal rule. Acute (first-phase) insulin response to glucose (AIRg) and acute C-peptide response to glucose (ACPRg) were calculated as the mean incremental response above baseline (average of −20 and −10 min) from samples drawn in the first 10 min of the clamp. Second-phase insulin response to glucose and second-phase C-peptide response to glucose were calculated as the mean incremental response in the last 40 min. We defined the maximum concentration (C_max) and T_max for insulin and C-peptide. We calculated insulin sensitivity index (ISI) as the mean glucose infusion rate in the last 40 min of hyperglycemic clamp divided by average plasma insulin in the same period (12). Glucagon level (AUC) was determined between 0 and 120 min.

Prehepatic insulin secretion rate (ISR) (pmol/min/m²) was determined by deconvolution of peripheral C-peptide concentrations using a two-compartment model of C-peptide kinetics and population-based C-peptide kinetic parameters (13,14). Basal insulin secretion (ISRb) was calculated as mean ISR (AUC/time) from −5 to 0 min. Absolute first-phase insulin secretion (ISR1abs) was calculated as mean ISR (AUC/time) between 0 and 8 min. Absolute second-phase insulin secretion (ISR2abs) was calculated as mean ISR (AUC/time) between 90 and 120 min. Incremental first-phase insulin secretion (ISR1inc) and incremental second-phase insulin secretion (ISR2inc) were defined as ISR1abs minus ISRb and ISR2abs minus ISRb, respectively. βCGS (slope of ISR vs. glucose) was calculated as ISR2inc divided by glucose change (steady-state glucose minus basal glucose) (15).

Data are summarized as mean ± SD or median (interquartile range [IQR]). Paired samples t test was used to compare differences for normally distributed data and Wilcoxon signed rank test for nonnormally distributed data between treatment conditions. Statistical analyses were conducted using R version 4.1.2 and SPSS version 26 (IBM Corporation) software. P < 0.05 was considered significant. No formal calculation of sample size was conducted for this proof-of-concept study. The sample size in each group was considered appropriate for the study objectives based on prior experience of glucose clamp studies of similar design. Adjustment for multiplicity was not applied in this proof-of-concept study.

Recombinant wild-type and three mutant human β-cell GKs (carried by the patients with GCK-MODY in this study) fused to glutathionyl-S-transferase (GST) tag were prepared by GenScript Biotech (Singapore) Ltd. as previously described (16). We measured GK activity spectrophotometrically (Varioskan Flash; Thermo Fisher Scientific) using an NADP⁺-coupled assay with glucose-6-phosphate dehydrogenase (16,17), with slight modifications. To evaluate the effects of dorzagliatin on GK activity in vitro, wild-type pancreatic GK and mutants were first incubated with 10 μmol/L dorzagliatin (cat. no. HY-109030; MedChemExpress) for 30 min. The same experimental procedures were repeated with calculation of kinetic constants and activity index (17). Kinetic parameters were generated by SigmaPlot version 14 software (Systat Software Inc.) (Supplementary Methods).

Deidentified data underlying the results reported in this article will be made available at least 12 months and <5 years after article publication, upon reasonable written request to the corresponding author. No applicable resources were generated or analyzed during the current study.

We enrolled 8 patients with GCK-MODY and 10 patients with recent-onset type 2 diabetes (Table 1), all of whom completed the study. The GCK-MODY group was younger (mean ± SD age 36.1 ± 6.4 vs. 50.2 ± 6.9 years) with lower BMI (23.9 ± 2.6 vs. 26.9 ± 2.2 kg/m²) than the type 2 diabetes group. HbA_1c (6.7 ± 0.3 vs. 6.9 ± 0.4% [49.6 ± 3.2 vs. 51.5 ± 4.4 mmol/mol]) and fasting plasma glucose (6.8 ± 0.5 vs. 7.0 ± 1.1 mmol/L) were similar between the two groups. The median diabetes duration in the latter group was 0.9 (IQR 0.6–1.4) years, with 70% of the participants treated with metformin. All participants with GCK-MODY were drug naive.

We identified the participants with GCK-MODY from three families. Five carried GCK heterozygous missense mutation c.1018 A>C (p.Ser340Arg); one carried GCK heterozygous missense mutation c.659G>A (p.Cys220Tyr), and two carried GCK heterozygous deletion mutation c.1132_1133del (p.Ala378CysfsTer80) (Supplementary Table 1). The pedigrees of the three families are shown in Supplementary Fig. 2. All participants with GCK-MODY were negative for anti-GAD antibodies. All 10 participants with type 2 diabetes were negative for mutations for GCK and 34 known genes associated with monogenic diabetes on the basis of targeted sequencing (18). None of the participants with GCK-MODY carried mutations of other forms of monogenic diabetes.

Basal blood glucose levels were lower after dorzagliatin administration than placebo in the GCK-MODY group (4.6 ± 0.6 vs. 5.4 ± 0.3 mmol/L, P = 0.010) at the start of the hyperglycemic clamp. Steady-state blood glucose concentrations were similar following dorzagliatin and placebo in the GCK-MODY group (11.9 ± 0.2 vs. 11.6 ± 0.6 mmol/L, P = 0.115) and type 2 diabetes group (11.9 ± 0.2 vs. 11.9 ± 0.3 mmol/L, P = 0.979) (Fig. 1A and E and Table 2).

In the GCK-MODY group, basal insulin secretion was similar following dorzagliatin compared with placebo (71.8 ± 44.4 vs. 57.5 ± 27.6 pmol/min/m², P = 0.276). AIRg (226.1 ± 64.3 vs. 179.4 ± 117.7 pmol/L, P = 0.164), ACPRg (675.5 ± 251.1 vs. 577.1 ± 363.0 pmol/L, P = 0.334), ISR1abs (443.3 ± 160.0 vs. 361.7 ± 210.3 pmol/min/m², P = 0.148), and ISR1inc (371.6 ± 141.0 vs. 304.2 ± 204.5 pmol/min/m², P = 0.216) were similar between dorzagliatin and placebo visits. During the second phase, dorzagliatin significantly increased ISR2abs (413.6 ± 85.1 vs. 271.7 ± 91.3 pmol/min/m², P = 0.002) and ISR2inc (341.8 ± 91.1 vs. 214.3 ± 74.4 pmol/min/m², P = 0.004) versus placebo (Fig. 1, Table 2, and Supplementary Fig. 3).

Figure 2A shows the ISR in participants with GCK-MODY with different GCK mutations. The carrier of the Cys220Tyr mutation appeared to be less responsive to dorzagliatin than the carriers of the Ser340Ag and Ala378CysfsTer80 mutations.

In the type 2 diabetes group, ISRb was higher with dorzagliatin versus placebo (102.3 ± 57.0 vs. 60.4 ± 41.8 pmol/min/m², P = 0.018). Unlike the GCK-MODY group, AIRg (−2.3 ± 22.6 vs. 31.5 ± 22.1 pmol/L, P = 0.006), ACPRg (48.0 ± 58.4 vs. 120.9 ± 66.4 pmol/L, P = 0.004), ISR1abs (109.6 ± 41.4 vs. 138.1 ± 59.4 pmol/min/m², P = 0.013), and ISR1inc (7.3 ± 30.1 vs. 77.7 ± 49.8 pmol/min/m², P = 0.005) were lower following dorzagliatin versus placebo. During the second phase, dorzagliatin numerically increased ISR2abs (296.3 ± 92.6 vs. 261.6 ± 101.0 pmol/min/m², P = 0.097), but differences were smaller compared with those in the GCK-MODY group (Fig. 1, Table 2, and Supplementary Fig. 3).

In both groups, the plots of ISR against blood glucose were shifted upward and leftward during dorzagliatin versus placebo administration (Fig. 1D and H). βCGS, expressed as slope of the ISR against blood glucose, was 1.5 times higher during dorzagliatin versus placebo in the GCK-MODY group (51.0 ± 20.6 vs. 33.4 ± 12.6 pmol/min/m² per mmol/L, P = 0.028) but remained unchanged in the type 2 diabetes group (27.7 ± 7.3 vs. 30.6 ± 12.8 pmol/min/m² per mmol/L, P = 0.295). Dorzagliatin did not affect the ISI estimated from glucose infusion rates during the hyperglycemic clamp in either group (Table 2).

Baseline plasma glucagon levels were 8.5 ± 3.7 pmol/L (dorzagliatin) and 7.9 ± 4.7 pmol/L (placebo) in the GCK-MODY group vs. 6.8 ± 4.0 pmol/L (dorzagliatin) and 7.5 ± 4.9 pmol/L (placebo) in the type 2 diabetes group. Plasma glucagon levels decreased progressively during the hyperglycemic clamps, with no difference between dorzagliatin and placebo in either group (Supplementary Fig. 4). The glucagon AUC was similar during dorzagliatin and placebo treatment in both groups.

There were no serious adverse events or deaths or adverse events that led to treatment discontinuation. Other nonserious adverse events are shown in Supplementary Table 2. One participant (D08) experienced mild hypoglycemia after dosing with dorzagliatin and before the start of the hyperglycemic clamp, which was corrected by a small intravenous bolus of 20% dextrose. Another participant (D07) reported one mild hypoglycemic event 8 days after the first dosing of placebo, which was deemed unlikely to be related.

Figure 2B shows the kinetic constants and relative activity index (I_a) of the proteins. Dorzagliatin increased I_a of wild-type GK by 50 times (from 1.00 ± 0.02 to 46.66 ± 8.77, P < 0.05). Of the mutant GK, Ser340Arg and Cys220Tyr had lower maximum velocity of phosphorylating glucose and higher half saturation concentration (S_0.5) than the wild-type GK, leading to decreased enzyme activity. S_0.5 of Ser340Arg was only mildly elevated (8.08 ± 0.50 mmol/L, P > 0.05), whereas S_0.5 of Cys220Tyr was 10-fold higher (89.07 ± 9.96 mmol/L, P < 0.05) compared with wild-type GST-GK (7.09 ± 0.10 mmol/L). Dorzagliatin rescued the enzyme activity of Ser340Arg comparable to that of wild-type GST-GK. For Cys220Tyr, dorzagliatin lowered the S_0.5 by 50%; however, the restored enzyme activity remained lower than the wild type without treatment (I_a 0.007 ± 0.001 vs. 1.00 ± 0.02). Dorzagliatin did not affect the maximum velocity of phosphorylating glucose or S_0.5 of ATP and had minimal effects on the cooperativity of glucose binding (Hill coefficient). For the frameshift mutation Ala378CysfsTer80, no activity was detected with or without dorzagliatin.

This study is the first to confirm that the molecular defect in GCK-MODY can be corrected by GKAs as a novel class of glucose-lowering drugs. Using glucose clamps, we demonstrated that dorzagliatin altered glucose sensitivity and shifted the ISR-glucose curve upward and leftward with improved insulin secretion, more so in heterozygous carriers of mutant GK than those with recent-onset type 2 diabetes. We further confirmed that dorzagliatin increased in vitro enzyme activity of wild-type GK and enhanced enzyme activity of two loss-of-function GK mutants (Ser340Arg and Cys220Tyr). Although dorzagliatin did not alter the activity of mutant GK Ala378CysfsTer80 in vitro, it improved β-cell function in its heterozygous carrier during the clamp study. These in vitro and in vivo data confirmed that dorzagliatin augmented activities of normal and mutant GK to restore the β-cell function in patients with GCK-MODY who carried one copy of mutant protein.

In our study, dorzagliatin increased second-phase ISR in GCK-MODY once the primary defect of βCGS was corrected. In vivo, dorzagliatin improved βCGS and second-phase ISR for all three mutant GKs, albeit with some heterogeneity. In vitro, the drug directly altered the kinetics of two of three mutant GKs. The p.Ser340Arg mutant is novel, although a different mutation in the same position has been reported (National Center for Biotechnology Information ClinVar RCV000029830.1) (19). Dorzagliatin improved the activity of GST-GK (p.Ser340Arg) to that of the wild type, and a significant improvement in second-phase ISR was also seen in vivo. For the Cys220Tyr mutant, the loss of cysteine in position 220 disrupts a potential disulphide bond for a stable conformation to interact with glucose (20). GST-GK (Cys220Tyr) exhibits a 10-fold lower affinity for glucose and increased degradation of the mutant protein in mouse models (21). Although dorzagliatin reduced S_0.5 by 50% in GST-GK (Cys220Tyr), the restored activity remained lower than the wild-type GK without treatment. p.Ala378CysfsTer80 is known and causes frameshift (22). In vitro, this mutant protein showed no activity with glucose, although in vivo, dorzagliatin improved βCGS by twofold and second-phase insulin secretion in carriers of p.Ala378CysfsTer80. We hypothesize that dorzagliatin might upregulate the activity of wild-type GK in these heterozygous carriers similar to the hypersensitivity to sulfonylureas in hepatic nuclear factor 1-α MODY (6). Thus, potentially, dorzagliatin can enhance ISR through direct action on selected GK mutants and/or through the wild-type GK to variable degrees, depending on both the mutation and β-cell reserve; however, the contribution of each is more difficult to dissect in human experiments.

In type 2 diabetes, dorzagliatin increased basal insulin secretion by twofold with paradoxically lower first-phase insulin compared with placebo. In a clinical trial involving participants with diet-controlled type 2 diabetes, a single dose of another dual-acting GKA (piragliatin) also increased basal C-peptide and insulin levels, but not postchallenge C-peptide levels, during a 75-g OGTT (23). In rodent clamp studies, GKAs suppressed endogenous glucose production and increased basal prehepatic insulin secretion (24). Guided by the T_max of dorzagliatin (11), participants received dorzagliatin 90 min before commencement of the clamp. During this postdose basal period, increased glycolysis by dorzagliatin might cause a sustained increase in the ATP/ADP ratio, resulting in a higher ISRb due to release from presynthesized insulin stored in intracellular vesicles as a readily releasable pool (25). With the commencement of the hyperglycemic clamp, the magnitude of first-phase ISR depended partly on the size of the readily releasable pool, which is maintained by an equilibrium between exocytosis and refilling. In GCK-MODY, an appropriate equilibrium between exocytosis and refilling is maintained, thereby preserving the first-phase response, while in type 2 diabetes, the already altered equilibrium may lead to excess exocytosis compared with refilling, resulting in a reduced first-phase ISR following dorzagliatin (26).

We only evaluated the effect of dorzagliatin following a single dose, and the effects of repeated dosing in the longer term and sustained glucose response in both participant groups remain unknown. Although some GKAs have been associated with declining efficacy with time (27), dorzagliatin exhibited no loss of glycemic efficacy in drug-naive or metformin-treated patients with type 2 diabetes in phase 3 clinical trials, which included an extension period of up to 52 weeks (2,3). Indeed, downregulation of β-cell GCK expression is a potential mechanism of impaired insulin secretion and glycemic control in type 2 diabetes, even though GK is functional (28). Of note, in type 2 diabetes GK expression was reduced by >60% compared with individuals with normal glucose tolerance (29). In diabetic mouse models, 1-month treatment with dorzagliatin restored hepatic GCK gene expression and GK protein compared with the untreated group (30). By reducing glucotoxicity, chronic administration of dorzagliatin may restore GK expression with improved β-cell function (3).

Consistent with physiological studies of other GKAs, dorzagliatin did not affect insulin sensitivity (23). Dorzagliatin did not have any additional effect on glucagon suppression by hyperglycemia in either participant group. A previous study demonstrated that individuals with GCK-MODY exhibit normal glucagon suppression in response to oral and intravenous glucose compared with healthy controls (31), which may explain the lack of effect of GKA. Glucagon levels at the end of clamp were higher in the type 2 diabetes group than the GCK-MODY group. Although α-cell GK may play a direct role in glucose-mediated glucagon release (32), long-standing alterations in α-cell bioenergetics may lead to glucagon dysregulation that may not be rescued by short-term GK activation. In a rat model of type 2 diabetes, dorzagliatin administration also did not significantly alter glucagon levels (30). The current experimental model did not allow examination of incretin or hepatic responses of dorzagliatin, which would require alternative study designs.

We used a double-blind, placebo-controlled, crossover hyperglycemic clamp study design to allow for a comprehensive assessment of basal, first-phase, and second-phase insulin secretion to provide a detailed estimation of β-cell function and glucose sensitivity. However, there are several limitations in this study. We did not include an active comparator, such as metformin or gliclazide, although it is well known that patients with GCK-MODY do not respond to conventional glucose-lowering drugs. Patients with GCK-MODY in our study also did not respond to metformin, gliclazide, or dipeptidyl peptidase 4 inhibitors clinically, as shown by continuous glucose monitoring (Supplementary Fig. 5). The GCK-MODY and type 2 diabetes groups were not directly comparable because they were not matched on age and BMI, and we cannot exclude the possibility that younger age and lower BMI might contribute to the higher ISR in the GCK-MODY group. On a few occasions, we had to administer dextrose to correct hypoglycemia in the postdose baseline period, which could have influenced preclamp blood glucose levels, although the steady-state glucose levels were similar in all experimental periods. We did not apply multiplicity statistical adjustments, and therefore, the results should be interpreted with appropriate caution. The sample size is relatively small because of difficulty in recruiting patients with GCK-MODY. Finally, we did not include a healthy control group, which may contribute to a better understanding of the characteristics of this agent. In a study of dorzagliatin in healthy subjects (11), a single dose of dorzagliatin ranging from 5 to 50 mg resulted in a dose-proportional lowering of fasting and postprandial glucose as well as increases in insulin release following a standardized meal. Nevertheless, the detailed effects of dorzagliatin on β-cell function in individuals with normal glucose tolerance will need to be investigated in future studies.

Because of their mild symptoms, treatment is not currently recommended for heterozygous carriers of inactivating GCK mutations (18). Homozygous carriers of GCK mutations present as permanent neonatal diabetes mellitus with a more severe phenotype and require insulin treatment from birth. Here, dorzagliatin may have utility, especially if the activity of the mutant proteins can be corrected by the drug. Formal evaluations of the safety and efficacy of dorzagliatin in these patient subgroups are warranted, including risks of hypoglycemia and lipid abnormalities. However, there is also clinical heterogeneity among heterozygous carriers of GCK mutations, with sporadic reports of more severe forms, for example, secondary to nonfunctional Gly261Arg enzyme with high postchallenge glucose values (33). Concomitant autoimmunity, insulin resistance, or other monogenic diabetes mutations could contribute to more severe phenotypes (34,35). In a study of 78 Japanese patients with GCK-MODY, up to 25% had concomitant insulin resistance. In this study, the adult patients with GCK-MODY also exhibited higher mean HbA_1c compared with children (7.8% vs. 7.3%) (61.8 mmol/mol vs. 56.3 mmol/mol) (36). Selected individuals with more severe phenotypes may benefit from dorzagliatin to reduce the glycemic burden that accumulates with advancing age. Single nucleotide polymorphisms of GCK and/or GCKR that encode GK regulatory protein (GKRP) are not uncommon in Chinese (37,38). Future studies are needed to examine whether these genetic variants might influence treatment response to dorzagliatin.

In conclusion, dorzagliatin directly increased enzyme activity of selected mutant GK and enhanced wild-type GK activity, thereby correcting the primary defect of glucose sensing in GCK-MODY. GK is the first step in triggering insulin secretion from pancreatic β-cells and hepatic glycogen production. Dorzagliatin increased pancreatic GK activity and improved β-cell function in GCK-MODY and to a lesser extent in type 2 diabetes. In patients with GCK-MODY, dorzagliatin as a GKA is an example of precision medicine in diabetes by rescuing the activity of their loss-of-function GCK variants.

Acknowledgments. The authors thank all staff and nurses of Phase 1 Clinical Trial Centre, The Chinese University of Hong Kong, for conducting and coordinating the trial. They especially thank all family members of the GCK-MODY group and the volunteers with type 2 diabetes who contributed valuable time and effort in helping with the trial.

Funding. This study was supported by a Hua Medicine Investigator Initiated Study (to J.C.N.C.) and The Chinese University of Hong Kong Direct Grant for Research (2020.231) (to E.C.). R.C.W.M. acknowledges support from the RGC Research Impact Fund (R4012-18) and a Croucher Foundation Senior Medical Research Fellowship.

The funder reviewed the protocol and final manuscript but had no role in the conceptualization, design, data collection, analysis, decision to publish, or preparation of the manuscript.

Duality of Interest. J.C.N.C. has received research grants and/or honoraria for consultancy and/or lectures from AstraZeneca, Bayer, Boehringer Ingelheim, Celltrion, Eli Lilly, Hua Medicine, Lee Powder, Merck Serono, Merck Sharp & Dohme, Pfizer, Servier, Sanofi, and Viatris; holds patents for using biomarkers to predict risks of diabetes and its complications; and is a cofounder of GemVCare, a biotechnology company partially supported by the Hong Kong Government startup fund. E.C. has received research grants and/or honoraria for lectures from Sanofi, Lee Powder, Medtronic Diabetes, and Novartis. A.M. has received financial support from Eli Lilly and is a consultant for Eli Lilly. C.K.P.L. and R.C.W.M. are cofounders of GemVCare, a technology startup initiated with support from the Hong Kong Government Innovation and Technology Commission and its Technology Start-up Support Scheme for Universities. L.C. is an affiliate of Hua Medicine. No other potential conflicts of interest relevant to this article were reported.

Author Contributions. E.C., K.W., C.K.P.L., S.T.F.T., B.F., E.P., and A.O.Y.L. collected the data. E.C., K.W., and J.C.N.C. drafted the manuscript. E.C. and J.C.N.C. conceived and designed the study. K.W., C.K.P.L., S.T.F.T., R.C.W.M., E.F., A.M., and L.C. analyzed and interpreted the data. All authors critically reviewed the manuscript and provided final approval of the version to be published. J.C.N.C. is the guarantor of the work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. Parts of this study were presented in oral abstract form at the 82nd Scientific Sessions of the American Diabetes Association, New Orleans, LA, in 3–7 June 2022.