Insulin has a narrow therapeutic index, reflected in a small margin between a dose that achieves good glycemic control and one that causes hypoglycemia. Once injected, the clearance of exogenous insulin is invariant regardless of blood glucose, aggravating the potential to cause hypoglycemia. We sought to create a “smart” insulin, one that can alter insulin clearance and hence insulin action in response to blood glucose, mitigating risk for hypoglycemia. The approach added saccharide units to insulin to create insulin analogs with affinity for both the insulin receptor (IR) and mannose receptor C-type 1 (MR), which functions to clear endogenous mannosylated proteins, a principle used to endow insulin analogs with glucose responsivity. Iteration of these efforts culminated in the discovery of MK-2640, and its in vitro and in vivo preclinical properties are detailed in this report. In glucose clamp experiments conducted in healthy dogs, as plasma glucose was lowered stepwise from 280 mg/dL to 80 mg/dL, progressively more MK-2640 was cleared via MR, reducing by ∼30% its availability for binding to the IR. In dose escalations studies in diabetic minipigs, a higher therapeutic index for MK-2640 (threefold) was observed versus regular insulin (1.3-fold).

Nearly a century has passed since the discovery of insulin, yet the remarkable success of insulin therapy has been tempered by the risk of iatrogenic hypoglycemia, which stands as a major barrier to the achievement of tight glycemic control (14). To mitigate risk for hypoglycemia, insulin analogs have been developed with improvements in pharmacokinetics (PKs) (57). However, efforts to improve insulin PKs do not enable exogenous insulin to autonomously modulate its action in the face of descending (or ascending) plasma glucose and thus do not change the intrinsically narrow therapeutic index of exogenously administered insulin. To address the latter, the notion of a glucose-responsive insulin (GRI) was proposed four decades ago (8). Attempts at creating a GRI have sought to incorporate insulin within a subcutaneous depot matrix containing glucose-sensitive chemical “triggers” that release insulin (912), but there is a substantial challenge in sufficiently modulating insulin release across the narrow physiological range of plasma glucose. An alternative strategy for a GRI, to exploit endogenous lectin–based clearance, was reported by Zion and Lancaster (13), and is the focus of this report.

Lectins recognize and bind carbohydrate domains of glycoproteins. Circulating and cell-based lectins function in immune surveillance and as clearance of glycoproteins (14,15). Mannose receptor C-type 1 (MR) is the prototypical member of the mannose receptor family of transmembrane lectins. Its main function is to recognize endogenous senescent proteins tagged for destruction and pathogens identified by their surface glycan and to deliver these for lysosomal degradation without eliciting an immune response or cytokine release (1619). MR is an abundant lectin in hepatic sinusoidal endothelial cells and on certain macrophages and dendritic cells, with a well-conserved homology across species (14). Glucose has a low affinity for MR but nevertheless can compete with binding of other ligands.

A concept of a GRI that could be engineered into an insulin analog by targeting MR-based clearance is illustrated in Fig. 1. This concept involves creating a chimeric insulin analog that undergoes a substantial fraction of its clearance through MR when plasma glucose is within the euglycemic or hypoglycemic range, thus lessening availability for interaction with the insulin receptor (IR), with the opposite direction effect at a progressively higher ambient glucose. We will describe our initial steps in creating a GRI using lectin-based clearance (J. Mu et al., unpublished observations), whereas the current report focuses on a single GRI, MK-2640. We describe the preclinical development of MK-2640, including dog and minipig in vivo studies designed to assess glucose responsivity.

Figure 1

A concept for GRI is illustrated that is based on adding saccharide modifications to the insulin molecule. The resulting analog has affinity for the IR as well as for an MR, which clears insulin via endocytosis and lysosomal degradation. Glucose competes for binding to the MR. At high ambient glucose, less of the analog is cleared by MR and a larger proportion becomes available for IR pharmacology; conversely, at low glucose, a proportionally larger fraction of the analog is cleared via MR, lowering availability for IR interaction. ICC, insulin-carbohydrate conjugate.

Figure 1

A concept for GRI is illustrated that is based on adding saccharide modifications to the insulin molecule. The resulting analog has affinity for the IR as well as for an MR, which clears insulin via endocytosis and lysosomal degradation. Glucose competes for binding to the MR. At high ambient glucose, less of the analog is cleared by MR and a larger proportion becomes available for IR pharmacology; conversely, at low glucose, a proportionally larger fraction of the analog is cleared via MR, lowering availability for IR interaction. ICC, insulin-carbohydrate conjugate.

Close modal

In Vitro Binding Assays

MK-2640 binding to IR was determined in a scintillation proximity assay with membranes prepared from CHO cells overexpressing human, minipig, or dog IR(B), and insulin as the competitive ligand (J. Mu et al., unpublished observations). MK-2640 direct binding to human or dog MR was assessed via surface plasmon resonance using His-tagged purified receptors immobilized onto a Biacore CM5 chip. To appraise glucose inhibition of MR binding, the surface plasmon resonance assay was modified to include varying concentrations of glucose or α-methylmannose (α-MM) in the presence of a fixed concentration of MK-2640, approximating its Kd value, to determine the glucose inhibitory IC50 for MK-2640 binding to human or dog MR. Competition binding assays for MR, DC-SIGN, and MBL were conducted using mannosylated-BSA, labeled with europium for fluorescent detection, and varying concentrations of MK-2640 to determine affinity for the human lectin receptors. An ex vivo assay of glucose inhibition of MK-2640 binding in the liver was conducted by perfusing the compound into the portal vein of mice with varying concentrations of glucose and then imaging liver sections with an anti-insulin antibody. Briefly, the portal vein of mice was cannulated for perfusion with MK-2640, and varying concentrations of glucose were added to the infusion buffer. Liver caudate lobe samples were taken and fixed (10% buffered formalin), and immunohistochemistry was performed with guinea pig anti-insulin antibody (Invitrogen #180067). Digital images were captured, and the percentage of immunoreactive cells was determined via instrument software (Membrane v9, Aperio Technologies).

In Vivo Evaluations of MK-2640

All animal procedures were reviewed and approved by the Merck Research Laboratories (Merck & Co., Kenilworth, NJ) Institutional Animal Care and Use Committee. The Guide for the Care and Use of Laboratory Animals was followed in the conduct of the animal studies. Veterinary care was given to any animals requiring medical attention.

Male Yucatan minipigs from the Sinclair Research Center, healthy (nondiabetic [ND]) or rendered type 1 diabetic (D) by alloxan injections, underwent placement of two jugular venous vascular access ports for administration of MK-2640 or recombinant human insulin (RHI; Humulin R, Lilly) intravenously (i.v.) and for blood sampling. The study used minipigs rendered diabetic by alloxan that attained plasma glucose levels of ∼300 mg/dL. RHI or MK-2640 was coadministered with PBS or α-MM (9.4 mg/kg/min), the latter a high-affinity ligand for MR that can act as a chemical blockade of MR (20). Insulin levels were measured by ELISA (RHI) or by liquid chromatography–mass spectrometry for MK-2640. To elucidate glucose-responsiveness of RHI or MK-2640, respective clearances were assessed in D minipigs and compared with corresponding values obtained in ND minipigs that had been coinfused with PBS or α-MM. To appraise therapeutic index, MK-2640 or RHI was administered subcutaneously (s.c.) to D minipigs with dose escalation (successive 30% dose increments), and plasma glucose monitoring for 8 h (AU480 Clinical Chemistry Analyzer, Beckman Coulter) until a dose that caused hypoglycemia was identified. Plasma epinephrine was assessed by liquid chromatography–mass spectrometry to monitor the hypoglycemic counterregulatory response.

MK-2640 was examined in healthy 4- to 6-year-old male beagle dogs with euglycemic and hyperglycemic clamps to examine its PK and pharmacodynamic (PD) effects across a range of steady-state plasma glucose concentrations. Dogs were fasted overnight before a clamp and had previously been prepared with dual-ported femoral artery and femoral vein cannulations, respectively, for sample collection and infusions. Animals received an infusion of somatostatin (0.8 µg/kg/min) during 6 target glucose levels (80, 120, 160, 200, 240, and 280 mg/dL) to suppress endogenous insulin secretion. Each dog completed all clamps. RHI at an infusion rate of 3 pmol/kg/min, was used as a comparator to MK-2640. Pilot studies found an infusion rate for MK-2640 of 45 pmol/kg/min achieved PD equipoise with RHI when clamps were conducted at 200 mg/dL, and these respective infusion rates were used in all clamps. PK readouts were RHI and MK-2640 concentrations and clearance. A change in MK-2640 clearance was interpreted as reflecting glucose responsive PKs. MK-2640 was tested for activation of cytokine release in the plasma of dogs receiving MK-2640 or RHI infusions. Cytokine levels (interleukin [IL]-6, IL-10, IL-8, IL-2, and tumor necrosis factor-α) were evaluated by ELISA (Meso Scale Diagnostics, Rockville, MD), and dog blood exposed ex vivo to lipopolysaccharide (10 pg/mL) was a positive control.

Statistical Analysis

Data analyses were performed in GraphPad Prism (GraphPad Software, San Diego, CA). Calculations of P values were based on ANOVA and the unpaired Student t test, whichever was applicable. Statistical significance was defined as P < 0.05.

Design and Synthesis of MK-2640

Chemistry efforts focused on conjugating RHI at the amino termini, including A1, B1, and the B29 Lys amino groups, individually or as bis-functionalization with linkers bearing mono-, di-, or trisaccharide units, as shown in Fig. 2A. Glucose, mannose, and fucose were selected for exploring the identification of potential lead GRI candidates, and through studies of the structure activity relationship, a lysine-based, nonsymmetric, mannose based–linker structure was identified that began to demonstrate modest glucose modulation of its PKs. This recognition also helped to define an appropriate range for respective affinities for IR and MR. We subsequently observed that the use of fucose in place of mannose increased MR binding affinity and with iterations led to the identification of MK-2640 as an analog with promising glucose responsiveness, and its structure is shown in Fig. 2A. Additional information on the chemistry effort can be accessed in a recently published patent application and references therein (21). To support the mechanism of action studies in minipigs, compound 2 (Fig. 2B) was synthesized by modifications at both A1 and B29 positions with morpholines replacing saccharide units that render it with similar IR affinity but greatly reduced MR affinity compared with MK-2640.

Figure 2

A: Chemical ligation strategy to synthesize insulin oligosaccharide conjugates is outlined, together with the specific structure of MK-2640. B: The structure of compound 2 (MK-2640 analog with A1 and B29 positions conjugated with moieties possessing non-MR binding morpholines instead of saccharide units). IUPAC, International Union of Pure and Applied Chemistry; NHS, N-hydroxysuccinimide.

Figure 2

A: Chemical ligation strategy to synthesize insulin oligosaccharide conjugates is outlined, together with the specific structure of MK-2640. B: The structure of compound 2 (MK-2640 analog with A1 and B29 positions conjugated with moieties possessing non-MR binding morpholines instead of saccharide units). IUPAC, International Union of Pure and Applied Chemistry; NHS, N-hydroxysuccinimide.

Close modal

PK Properties of MK-2640

The PK properties of MK-2640 were evaluated in beagle dogs and Yucatan minipigs after i.v. and s.c. dosing. As a preliminary formulation effort, directed at achieving drug solubility, MK-2640 was formulated in sodium phosphate (26.5 mmol/L) buffer (pH = 7.4) containing sodium chloride (87 mmol/L) and phenol (21 mmol/L). Dogs were dosed i.v. at 2.5 µg/kg and s.c. at 15 µg/kg. Minipigs were dosed i.v. at 5 µg/kg and s.c. at 45 µg/kg. After i.v. dosing, MK-2640 exhibited moderate to high systemic clearance in dogs and minipigs (8 and 39 mL/min/kg, respectively). The steady state volume of distribution was low (∼0.04–0.2 L/kg), and the elimination half-life was short (3–14 min) in these species. After s.c. administration of MK-2640, absorption was faster in the dogs than in the minipigs (tmax 18 min vs. 36 min), and the bioavailability varied between 26% and 44%. The biodistribution of MK-2640 was examined in healthy rats using s.c. dosing of [3H]MK-2640, and this showed significant amounts of [3H]MK-2640–related radioactivity detected in liver, kidney, muscle, and fat.

Targeting of MR

The binding affinity of MK-2640 for the IR, as presented in Table 1, was substantially lower than RHI (∼4% in comparison), which suggests that the potential for full clinical development of MK-2640 might be limited due to low potency. However, the potential for glucose responsiveness was regarded as important to interrogate in vivo. In vitro, at the highest concentrations of MK-2640 examined, the compound completely blocked binding of native insulin. Binding to human MR was similar to that observed for the minipig and dog MR, as reported in Table 2.

Table 1

Cell membrane IR-binding affinities

IR binding
Human
Minipig
Dog
IC50SDIC50SDIC50SD
(nmol/L)(nmol/L)(nmol/L)
RHI 0.48 1.5 0.39 1.8 0.44 1.3 
MK-2640 7.0 2.1 7.4 1.2 8.5 1.1 
IR binding
Human
Minipig
Dog
IC50SDIC50SDIC50SD
(nmol/L)(nmol/L)(nmol/L)
RHI 0.48 1.5 0.39 1.8 0.44 1.3 
MK-2640 7.0 2.1 7.4 1.2 8.5 1.1 

Geometric mean IC50 and geometric SD are from four or more individual MK-2640 titrations for all species.

Compound 2 is an analog of MK-2640 wherein the saccharides are replaced by morpholino groups. The IR human binding activity of compound 2 was 5.7 nmol/L.

Table 2

MR-binding affinities

MR binding (Biacore)
MR binding (DELFIA)
Human
Dog
Human
Minipig
Dog
Kd (nmol/L) SDKd (nmol/L) SDIC50 (nmol/L) SDIC50 (nmol/L) SDIC50 (nmol/L) SD
MK-2640 3.4 1.4 2.7 1.3 28 1.2 23 1.3 16 1.2 
MR binding (Biacore)
MR binding (DELFIA)
Human
Dog
Human
Minipig
Dog
Kd (nmol/L) SDKd (nmol/L) SDIC50 (nmol/L) SDIC50 (nmol/L) SDIC50 (nmol/L) SD
MK-2640 3.4 1.4 2.7 1.3 28 1.2 23 1.3 16 1.2 

Geometric mean Kd and IC50 and geometric SD are from four or more individual MK-2640 titrations for all species. Compound 2 was inactive in the human IR binding assay. DELFIA, dissociation-enhanced lanthanide fluorescent immunoassay.

MK-2640 did not bind to MBL at the highest concentration tested (10 μmol/L) and had relatively weak affinity for DC-SIGN, with an IC50 of 1,800 nmol/L. Consistent with these findings, MK-2640 did not evoke cytokine release from human differentiated macrophages (Supplementary Fig. 1). By using increasing concentrations of glucose, as shown in Fig. 3A, the ability of glucose to compete with the binding of MK-2640 to MR was assessed in vitro, and an IC50 of 8 mmol/L glucose was determined; a corresponding value for a “high-affinity” MR–binding small molecule, α-MM, is 0.62 mmol/L. An ex vivo mouse liver perfusion model was used to assess whether glucose affected partitioning of selected analogs between IR and MR in the presence of both receptors. As shown in Fig. 3B, a large fraction of MK-2640 was taken up by MR at a low glucose concentration, and this fraction progressively declined as the glucose concentration was raised to hyperglycemia, with an IC50 value of ∼9 mmol/L glucose, quite similar to the IC50 estimated from in vitro binding experiments using immobilized MR (Fig. 3B).

Figure 3

Glucose modulation of MK-2640 binding to MR. A: Data from an MR Biacore binding assay are shown in which titrating glucose concentrations are used in the constant presence of 4 nmol/L MK-2640 (∼Kd concentration added to the running buffer). A glucose IC50 of ∼8 mmol/L was determined (compared with IC50 ∼0.62 mmol/L for α-MM). B: Ex vivo mouse liver perfusion assay where an increasing concentration of glucose was added to the buffer containing a fixed concentration of MK-2640. The MK-2640 taken up by the liver was quantified by immunohistochemistry using an anti-insulin antibody capable of detecting MK-2640. IOC, insulin oligosaccharide conjugate.

Figure 3

Glucose modulation of MK-2640 binding to MR. A: Data from an MR Biacore binding assay are shown in which titrating glucose concentrations are used in the constant presence of 4 nmol/L MK-2640 (∼Kd concentration added to the running buffer). A glucose IC50 of ∼8 mmol/L was determined (compared with IC50 ∼0.62 mmol/L for α-MM). B: Ex vivo mouse liver perfusion assay where an increasing concentration of glucose was added to the buffer containing a fixed concentration of MK-2640. The MK-2640 taken up by the liver was quantified by immunohistochemistry using an anti-insulin antibody capable of detecting MK-2640. IOC, insulin oligosaccharide conjugate.

Close modal

Glucose Responsiveness in Minipigs

MK-2640 PK was evaluated in ND and D Yucatan minipigs after i.v. dosing in the absence and presence of coinfused α-MM. The i.v. administration of RHI led to a short-lived fall of plasma glucose; its PKs and PDs were unaffected by α-MM, as shown in Fig. 4A and C. The PKs and PDs of MK-2640 in ND minipigs were strongly affected by α-MM coinfusion. A dose of MK-2640 (0.35 nmol/kg) that had rapid clearance and only a modest glucose-lowering effect in ND minipigs when administered alone produced transient hypoglycemia in the presence of α-MM (Fig. 4B), because its clearance was markedly protracted (Fig. 4D). The MK-2640 PK differences observed with and without α-MM served to demarcate potential boundaries of a corresponding maximal glucose responsiveness because these PK differences delineate the contribution of MR-mediated clearance (at euglycemia). Glucose-responsive PKs were then explored by giving RHI and MK-2640, each as an i.v. bolus to D minipigs. MK-2640 has been shown to dose-dependently lower glucose in D minipigs when administered i.v. (see Supplementary Fig. 2), and a dose (0.35 nmol/kg) with minimal glucose-lowering was used to evaluate its PKs during stable hyperglycemia. As shown for RHI (Fig. 4C) and MK-2640 (Fig. 4D), hyperglycemia in D minipigs did not change clearance of RHI versus ND, whereas the clearance for MK-2640 in D minipigs was decreased. This difference in clearance of MK-2640 in D versus ND minipigs was interpreted as highly similar to the PKs change observed with blockade of MR clearance by α-MM in ND minipigs. Additional studies demonstrated that α-MM–induced PK and PD changes for MK-2640 (Fig. 4B) cannot be ascribed to the reduced IR potency of MK-2640 per se because compound 2 (0.69 nmol/L i.v. dose), with comparable IR potency as MK-2640 but minimal MR binding affinity, demonstrated negligible PK differences when dosed in ND minipigs with versus without α-MM.

Figure 4

PDs and PKs after an i.v. bolus of RHI (0.17 nmol/kg) (A and C) and MK-2640 (0.35 nmol/kg) (B and D) given to ND and D minipigs with and without coinfusion of α-MM (ND only), which provides a short-term chemical blockade of MK-2640 clearance by MR. A and B: The effect of the two insulins is shown in ND minipigs with and without α-MM. C and D: The PK differences and the effects of α-MM in ND and D minipigs are compared. For panels AD, data is presented as mean ± SEM. Areas under the curve0-∞ for the different MK-2640 groups are indicated in panel D (as mean ± SD).

Figure 4

PDs and PKs after an i.v. bolus of RHI (0.17 nmol/kg) (A and C) and MK-2640 (0.35 nmol/kg) (B and D) given to ND and D minipigs with and without coinfusion of α-MM (ND only), which provides a short-term chemical blockade of MK-2640 clearance by MR. A and B: The effect of the two insulins is shown in ND minipigs with and without α-MM. C and D: The PK differences and the effects of α-MM in ND and D minipigs are compared. For panels AD, data is presented as mean ± SEM. Areas under the curve0-∞ for the different MK-2640 groups are indicated in panel D (as mean ± SD).

Close modal

Glucose Clamps

To assess glucose responsiveness of MK-2640, glucose clamp studies were performed in healthy dogs clamped at target plasma glucose concentrations of 80, 120, 160, 200, 240, and 280 mg/dL, one level per study, with complete crossover among dogs. Doses of MK-2640 (45 pmol/kg/min) and RHI (3 pmol/kg/min) were used that required similar glucose infusion rate (GIR) during clamps conducted at 200 mg/dL (11 ± 1 vs. 10 ± 1 mg/kg/min, respectively). These high rates of matched GIR, approximately fivefold above fasting rates of glucose flux, denote stimulation of glucose utilization within peripheral tissues consistent with the rat biodistribution data. Stepwise increases in plasma concentration of MK-2640 and corresponding decreases in clearance were observed with increased plasma glucose during clamps. The change in PKs of ∼30% across this glycemic range is shown in Fig. 5A. RHI plasma concentrations remained unaffected by glycemic level, and RHI clearance (23 ± 3 mL/kg/min) remained constant (Fig. 5B). For MK-2640, the reduction of GIR required to maintain target levels of glycemia at less than 200 mg/dL declined to a greater extent than in the corresponding clamps conducted with RHI. At a plasma glucose of 80 mg/dL, the steady state GIR was 4 ± 1 mg/kg/min for RHI and 2 ± 1 mg/kg/min for MK-2640 (P < 0.05). Two-hour infusions of MK-2640 did not evoke changes in cytokines and were not different from the responses observed with RHI, as shown in Supplementary Fig. 3.

Figure 5

Plasma drug concentrations for infused insulins were measured during steady-state conditions of a glucose clamp procedure performed in healthy dogs using a constant rate of infusion of 45 pmol/kg/min for MK-2640 (A) and an infusion of 3 pmol/kg/min for RHI (B). The target plasma glucose concentrations for each of the clamp studies are shown on the x-axis. A crossover study design was used, by which dogs participated in each of the clamps, and a glucose-dependent increase in MK-2640 plasma concentration was observed, consistent with a glucose-responsive modulation of its clearance, whereas no effect on the clearance of RHI was observed. Data are mean ± SEM.

Figure 5

Plasma drug concentrations for infused insulins were measured during steady-state conditions of a glucose clamp procedure performed in healthy dogs using a constant rate of infusion of 45 pmol/kg/min for MK-2640 (A) and an infusion of 3 pmol/kg/min for RHI (B). The target plasma glucose concentrations for each of the clamp studies are shown on the x-axis. A crossover study design was used, by which dogs participated in each of the clamps, and a glucose-dependent increase in MK-2640 plasma concentration was observed, consistent with a glucose-responsive modulation of its clearance, whereas no effect on the clearance of RHI was observed. Data are mean ± SEM.

Close modal

Partitioning of MK-2640 to Uptake by MR in Dogs

When compared during clamp studies conducted at a plasma glucose of 200 mg/dL, the infusion rate of MK-2640 (45 pmol/kg/min) needed to achieve PD equipoise with a 3 pmol/kg/min infusion of RHI was 15-fold greater. This difference can partly be attributed to the lower in vitro potency of MK-2640 for the IR. However, it is potentially important to take into account the percentage of MK-2640 partitioned to degradation via MR-mediated uptake. Clamp studies were conducted in dogs with and without a concomitant infusion of α-MM (9.65 mg/kg/min). At a plasma glucose of 80 mg/dL, infusion of α-MM reduced the clearance of MK-2640 by 81%, delineating that a high fraction of its clearance was via MR, leaving the remaining 19% to be cleared by other pathways, presumably by IR. At a plasma glucose of 300 mg/dL, the overall clearance of MK-2640 was reduced by ∼33% compared with euglycemic conditions, and with infusion of α-MM during hyperglycemia, clearance by MR still accounted for a large fraction (∼66%) of its clearance.

Dose Escalation to Hypoglycemia

Dose escalations (30% successive increases) with s.c. administration of MK-2640 and RHI in D minipigs were performed to determine the effect of upward titrations of dose to lower plasma glucose and to thereby identify a dose that caused hypoglycemia. To estimate the therapeutic index of RHI and MK-2640, the ratio of a dose that caused hypoglycemia (defined as a value of 55 mg/dL), relative to the minimally efficacious dose (MED), was calculated; MED was defined as the dose at which at least 50% of the animals reach glucose levels <120 mg/dL. As shown in Fig. 6A, s.c. administration of RHI led to a rapid decrease in plasma glucose levels, with a MED of 0.41 nmol/kg. Further increases above the MED for RHI resulted in a fall of blood glucose levels into the hypoglycemic range. At a dose of RHI of 0.53 nmol/kg, a robust counterregulatory epinephrine response occurred, with a 150% increase over baseline values observed at hypoglycemia (Fig. 6C); this RHI dose was only a 1.3-fold increment over the MED. MK-2640 caused a dose-dependent decrease in plasma glucose with an MED of 2.1 nmol/kg (Fig. 6B), and hypoglycemia caused by MK-2640 was not observed until the 8.0 nmol/kg dose, a nearly fourfold therapeutic index. A counterregulatory response of epinephrine was, however, observed with an MK-2640 dose of 6.1 nmol/kg and was more pronounced at the 8.0 nmol/kg dose (Fig. 6D). By these criteria, the therapeutic index for MK-2640 can be estimated as 3:1 relative to its MED.

Figure 6

Plasma glucose levels (A and B) and changes in plasma epinephrine levels (C and D) in D minipigs after s.c. administration of RHI (A and C) and MK-2640 (B and D) in escalating doses of a 30% increment over the preceding dose until the dose yielding a hypoglycemic response was observed. The shadowed area in A and B (55–120 mg/dL) defines the euglycemia range for the minipigs. The shadowed areas in C and D represent the normal range of variance for plasma concentrations of epinephrine under resting conditions in the D minipigs. Data are mean ± SEM.

Figure 6

Plasma glucose levels (A and B) and changes in plasma epinephrine levels (C and D) in D minipigs after s.c. administration of RHI (A and C) and MK-2640 (B and D) in escalating doses of a 30% increment over the preceding dose until the dose yielding a hypoglycemic response was observed. The shadowed area in A and B (55–120 mg/dL) defines the euglycemia range for the minipigs. The shadowed areas in C and D represent the normal range of variance for plasma concentrations of epinephrine under resting conditions in the D minipigs. Data are mean ± SEM.

Close modal

Subchronic Dosing of MK-2640 in D Minipigs

Proof of concept that sustained insulin therapy can maintain reduction of hyperglycemia is well established. However, given the novelty of a mannosylated insulin such as MK-2640, subchronic studies in D minipigs were undertaken to assess stability of glycemic control. Thrice-daily injections of MK-2640 or RHI were administered to D minipigs for 2 weeks after animals were withdrawn from their usual long-acting insulin therapy. Repeated dosing of MK-2640 achieved control of hyperglycemia during the 2 weeks of dosing that was comparable to that attained with RHI, as shown in Supplementary Fig. 4. Repeated dosing of MK-2640 was well tolerated, did not cause local reactions at injection sites, and did not elicit the generation of anti-drug antibodies or anti-insulin neutralizing antibodies (Supplementary Table 1).

Formulation Efforts to Achieve a Basal Insulin Profile

The reduced in vitro potency of MK-2640 and its consequent relatively slow onset of action, even when administered i.v., fit a profile that seemed better suited as a long-acting basal insulin, rather than as a rapid-acting insulin, because the latter requires high in vitro potency. However, to achieve a sustained PK profile, it was necessary to advance beyond the preliminary formulation used in the PK and PD studies described above and explore development of a formulation of MK-2640 that could manifest slow absorption kinetics over hours rather than minutes. Exploratory formulation work was begun to assess whether slow absorption kinetics could be achieved for MK-2640. The preliminary results are shown in Supplementary Fig. 5, suggesting that with further refinement, a basal insulin preparation could be achieved for MK-2640 or other similar GRI candidates.

The synthesis of MK-2640 was undertaken in an effort to create an analog with a capacity to alter its PKs in a glucose-responsive manner and hence its availability for insulin action. The innovation was based on creating an insulin chimera by attaching saccharides to native insulin that would imbue capacity to bind to the lectin receptor MR yet retain capacity for alternatively binding to the IR. Upon binding to MR, the insulin analog MK-2640, like other ligands for MR, is taken up and degraded in lysosomes. This fate precludes interaction with the IR. Glucose responsiveness does not ensue per se from a capacity for binding to MR; rather, MR binding opens a pathway of insulin egress that does not evoke insulin action. Creating glucose responsiveness required that the interaction with MR could in turn be modulated by the level of plasma glucose. MK-2640 binding by MR in vitro is increased at low concentrations of glucose and vice versa. Liver perfusion studies also revealed that uptake by the MR pathway is modulated by ambient glucose in a linear response and with an IC50 of ∼9 mmol/L glucose very similar to that identified from in vitro binding studies. The results of these assays suggested that the clearance and degradation of MK-2640 by the MR pathway could have potential to be the greatest when ambient glucose is reduced, when it will be desirable to lower circulating insulin and thereby to hopefully mitigate risk for insulin-induced hypoglycemia.

From the outset of the protein engineering effort, the balance of the affinity for IR relative to MR would clearly be an essential parameter guiding construction of GRI candidate molecules. Attachment of saccharides to the insulin backbone enabled interaction with MR, and with iterations of saccharide selection, this interaction with MR could be tuned to respond to ambient glucose. Attachment of saccharides to the insulin backbone, the site of attachments (e.g., A1, B1, and/or B29 of insulin), and the length and composition of the linker joining a saccharide to insulin also affected analog potency on the IR (21). MK-2640 was identified as an analog that might have a desirable balance of respective binding affinities for IR and MR, and the glucose responsiveness of MK-2640 derives from modulation of its PKs because changes in ambient glucose do not modify the intrinsic potency of MK-2640 for the IR. The diminished in vitro potency of MK-2640 for the IR likely would position this or similar GRI analogs to find usefulness as a basal rather than as a rapid-acting insulin because the latter needs full in vitro potency so that its onset of action is rapid. Although the reduced IR potency of MK-2640 positions it as a potential basal insulin, additional effort to achieve a formulation that would slow its absorption kinetics would ultimately need to be achieved. In the current report, preliminary formulations efforts with MK-2640 indicated that slowing its absorption kinetics might be feasible, but the in vivo data presented for MK-2640 in dogs and minipigs, whether administered s.c. or by i.v. injection, used a simpler “fit for purpose” formulation that achieved good solubility of the analog and that was associated with relatively prompt absorption kinetics after s.c. administration.

The hypothesis that MK-2640 might have a desirable balance of respective binding affinities for IR and MR was tested in the dog and minipig, two preclinical species commonly used in the development of novel insulins. In both species, MK-2640 was shown to demonstrate glucose-responsive PKs and PDs. The MK-2640 infusion studies conducted in healthy dogs, using glucose clamp methods conducted across a range of target glucose concentrations, focused on PK changes and demonstrated that ambient glucose modulates in a linear manner the clearance of MK-2640, increasing its clearance at lower plasma glucose. Collateral studies conducted during coinfusion of α-MM, a high-affinity ligand for MR that provides chemical blockade of MK-2640 clearance by MR, revealed that at euglycemia, ∼80% of MK-2640 clearance is accounted for by MR-based clearance. Thus, a substantial portion of the MK-2640 dose was cleared via MR in the euglycemic clamp. On one hand, the considerable flux of MK-2640 through MR offers the potential for meaningful plasma PK shifts upon glucose modulation of MR binding. On the other hand, whether it is practical to have an insulin analog that undergoes this proportion of degradation independent of evoking insulin action and whether it is desirable to chronically engage the MR pathway to this extent by a drug administered daily clearly will require more investigation.

Glucose responsiveness was also clearly revealed in the studies using MK-2640 in ND and alloxan-induced D minipigs. The clearance of MK-2640 in ND minipigs was greatly impeded by an infusion of α-MM, a finding recapitulated in the setting of marked hyperglycemia of D minipigs. Importantly, dose escalation studies in D minipigs suggested a safer therapeutic index for MK-2640 compared with regular insulin. The working definition of therapeutic index for insulin used in our studies was simple: it was to compare the dose that induced hypoglycemia relative to one that reduced fasting hyperglycemia to a desirable target of 120 mg/dL. In the comparison of MK-2640 to regular insulin, the MK-2640 dose could be increased by threefold above the MED before hypoglycemia ensued or hypoglycemic counterregulation was evoked. For regular insulin, the dose escalation was an order of magnitude smaller, a 30% increase above the MED caused hypoglycemia and a counterregulatory response.

Insulin-induced hypoglycemia is the most serious acute complication of using this medicine (2,3). Innovations in insulin chemistry have yielded basal insulin preparations with more consistent duration of the absorptive phase and flatter PK profiles, while efforts on prandial insulin continue to strive for rapid onset of action (6,7). Yet, these insulin analogs continue to manifest a narrow therapeutic index and do not per se contain a capacity to modify insulin action once the compound has been administered. The aspiration of a GRI is to engineer into an insulin preparation a “postinjection” flexibility that can modulate insulin availability based on changing needs, as dictated by changes in blood glucose. Some of the approaches that have been explored or are still being explored for engineering GRI center on introducing glucose-dependent flexibility in the release of insulin from its injected depot (9,10,12). The approach we have undertaken with the compound MK-2640 is to introduce a capacity for glucose responsiveness onto the backbone of native insulin itself to enable its uptake and degradation by the MR pathway. In this specific regard, it is interesting to consider that a major site for expression of MR is on hepatic sinusoidal endothelial cells that are closely adjacent to underlying hepatocytes, cell types separated narrowly by the space of Disse (14,15). More studies are needed to understand not only the systemic changes in the PKs and PDs of a glucose-responsive analog such as MK-2640 but also the tissue- and organ-specific contributions to glucose responsiveness, notably the contribution of the hepatic bed. Although a key site of drug disposal for MK-2640 within hepatic sinusoidal endothelial cells is likely due to the high expression of MR in these cells, biodistribution studies and relatively high rates of GIR stimulated during dog clamp studies indicate that MK-2640 is not a “liver-selective” insulin analog per se.

The intent of the reported studies is to focus on proof of pharmacology that engineering a novel GRI analog using a strategy of lectin-based glucose tunable clearance is feasible. Across the range of plasma glucose from 80 to 300 mg/dL, the clearance of MK-2640 changed by 30%, which is unprecedented, but how much glucose-based PK modulation of a GRI analog must be achieved to attenuate risk for hypoglycemia is unknown. In the D minipig studies of escalating dose until hypoglycemia ensued, a substantially greater therapeutic index was observed for MK-2640 than for RHI (threefold vs. 1.3-fold, respectively). In general, the greater the amount of glucose-responsive PK change and the greater the therapeutic index, the greater would seem a potential to mitigate the risk for hypoglycemia. Whether the degree of glucose responsiveness achieved by MK-2640 in preclinical studies has sufficient potential to attain a meaningful reduction risk for hypoglycemia remains unknown.

In summary, proof of pharmacology preclinical studies, together with relevant in vitro assays, are presented that demonstrate preliminary feasibility in engineering glucose responsivity onto native insulin. The PKs of MK-2640 can change by nearly one-third across a physiologically relevant range of plasma glucose values that would be experienced by an individual with insulin-requiring diabetes. The glucose-dependent decrease in PKs at euglycemia compared with hyperglycemia was mediated by increases in the clearance of MK-2640 by MR and was associated with reduced insulin action on glucose metabolism. The first locus of control in insulin therapy is cognitive and resides with a physician and patient selecting an appropriate dosage, and the second locus of control with currently available insulin resides in its absorption kinetics because elimination kinetics are invariant. We hope that by adding to these, a third modulus of control, that of glucose-responsive variance of insulin elimination kinetics once a GRI is already circulating in plasma, that a potential for increased efficacy and safety can be introduced into insulin therapy.

Acknowledgments. The authors thank many Merck colleagues for their contributions to this project, including Raul Camacho, Carlos G. Rodriguez, and Joel Mane for technical and scientific support during in vivo studies, Xiaoping Zhang and Steven Williams for IR-binding data, and Xun Shen for the MR-binding data. The authors wish to acknowledge Hsuan-shen Chen, Huaibing He, Tina Santos, Ling Xu, Xiaofang Li, Xinchun Tong, and Bernard Choi, of the Discovery Bioanalytics Group, Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, Rahway, NJ, for the quantitative analysis of MK-2640, insulin, and epinephrine. The authors also acknowledge the valuable contributions of Merck colleagues Nancy Thornberry, Natarajan Sethumaran, Michael Meehl, and Gus Gustafson for early contributions to the project, Paul Carrington for editorial comments, and Alan Cherrington, of Vanderbilt University, for insightful discussions throughout the course of this project.

Duality of Interest. All of the authors were full-time employees of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, and held stock or stock options in the company during the conduct of these studies. Merck Research Laboratories was the sponsor of this research. No other potential conflicts of interest relevant to this article were reported.

Author Contributions. N.C.K., S.L., T.K., M.v.H., J.M., V.R., M.E., R.P.N., and D.E.K. interpreted the data. N.C.K., T.K., M.v.H., J.M., E.C.-J., V.R., S.S., and D.E.K. designed the studies conducted at Merck Research Laboratories. N.C.K., L.Y., T.K., M.v.H., J.M., M.W., G.D., Y.C., Y.Z., E.C.-J., V.R., P.Z., S.S., A.O., F.L., S.C.S., and W.S. researched data. N.C.K., J.M., R.P.N., and D.E.K. wrote the manuscript. S.L., P.H., S.S., V.A., J.L.D., and R.P.N. led the medicinal chemistry research that discovered and synthesized MK-2640. R.P.N. and D.E.K. are the guarantors of this work and as such had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. Parts of this study were presented in abstract form at the 25th American Peptide Symposium, Whistler, BC, Canada, 17–22 June 2017.

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