A selective nonpeptide somatostatin receptor 5 agonist effectively decreases insulin secretion in hyperinsulinism

Abstract

Congenital hyperinsulinism (HI), a beta cell disorder most commonly caused by inactivating mutations of beta cell K_ATP channels, results in dysregulated insulin secretion and persistent hypoglycemia. Children with K_ATP-HI are unresponsive to diazoxide, the only FDA-approved drug for HI, and utility of octreotide, the second-line therapy, is limited because of poor efficacy, desensitization, and somatostatin receptor type 2 (SST2)-mediated side effects. Selective targeting of SST5, an SST receptor associated with potent insulin secretion suppression, presents a new avenue for HI therapy. Here, we determined that CRN02481, a highly selective nonpeptide SST5 agonist, significantly decreased basal and amino acid–stimulated insulin secretion in both Sur1^−/− (a model for K_ATP-HI) and wild-type mouse islets. Oral administration of CRN02481 significantly increased fasting glucose and prevented fasting hypoglycemia compared to vehicle in Sur1^−/− mice. During a glucose tolerance test, CRN02481 significantly increased glucose excursion in both WT and Sur1^−/− mice compared to the control. CRN02481 also reduced glucose- and tolbutamide-stimulated insulin secretion from healthy, control human islets similar to the effects observed with SS14 and peptide somatostatin analogs. Moreover, CRN02481 significantly decreased glucose- and amino acid-stimulated insulin secretion in islets from two infants with K_ATP-HI and one with Beckwith-Weideman Syndrome-HI. Taken together, these data demonstrate that a potent and selective SST5 agonist effectively prevents fasting hypoglycemia and suppresses insulin secretion not only in a K_ATP-HI mouse model but also in healthy human islets and islets from HI patients.

Congenital hyperinsulinism (HI) is a beta cell disorder that results in dysregulated insulin secretion and persistent hypoglycemia. HI is the most common cause of persistent hypoglycemia in neonates, infants, and children, and it is associated with a high risk of serious complications such as seizures, developmental and behavioral problems, and intellectual disabilities (1, 2). Up to 14 genetic loci have been associated with HI; however, the most common causes are inactivating mutations in the genes encoding the ATP-sensitive potassium channel (K_ATP-HI), accounting for up to 57% of cases followed at our Center located in Philadelphia, Pennsylvania (3), which is consistent with the experience of other HI Centers (4, 5). Currently, available treatments for K_ATP-HI are extremely limited. The only FDA-approved drug for the treatment of HI is the K_ATP channel activator, diazoxide. Consequently, due to the mutations that alter the functionality or expression of K_ATP channels resulting in their HI, children with K_ATP-HI are typically unresponsive to this therapy. Octreotide, a somatostatin analogue, is used as a second-line therapy. However, due to limited efficacy, tachyphylaxis, and side effects, such as gastrointestinal discomfort, thrombosis, and necrotizing enterocolitis, its use is limited, particularly in neonates (6, 7). Unfortunately, due to the dearth of therapies and the limitations of available treatments, most infants with K_ATP-HI require a pancreatectomy for intractable hypoglycemia. Infants with the focal form of the disease, which account for approximately 50% of cases, can often be cured by resection of the lesion by an experienced surgical team, however, for cases with diffuse disease, pancreatectomy is only palliative with 50% of patients requiring additional therapy for persistent hypoglycemia (8, 9). As a result, there is a dire need for new and better therapies for infants with K_ATP-HI.

Somatostatin analogs (SSAs) are an attractive option for HI treatment due to their ability to effectively inhibit insulin secretion. Somatostatin achieves its inhibitory effects on pancreatic hormone secretion through interaction with somatostatin receptors (SSTs), which belong to the superfamily of G protein–coupled receptors (GPCR) (10, 11). The expression of SSTs in the mammalian pancreas has been an area of significant interest. RT-PCR analysis of the rodent pancreas has confirmed gene expression of SST1, 2, 3, and 5 but not SST4 (12). In addition, immunostaining of the rodent pancreas has identified the expression of SST2 on alpha cells, while beta and delta cells express both SST2 and SST5 (13, 14, 15, 16, 17, 18). The use of knockout mouse models and receptor-specific analogs established that SST2 modulates the suppression of both insulin and glucagon, whereas SST5 principally regulates insulin release (19, 20, 21, 22). Both pancreatic alpha and beta cells in humans exhibit high expression of SST2, while beta cells additionally express SST1 and 5 (10, 13, 23, 24, 25, 26). In contrast to rodent models, SST2 activation in human islets inhibits glucagon secretion. and SST1, 2, and 5 receptors participate in the regulation of insulin secretion (10, 27, 28, 29). This unique network of somatostatin receptor expression and regulation of glucose homeostasis provides potential targets for receptor-specific strategies for the treatment of HI. Octreotide, used off-label in the treatment of HI, has an affinity for SST2; thus, some of its observed lack of effectiveness may be explained by a weaker affinity for beta-cell specific SSTs and potentially counterproductive on-target effects such as glucagon suppression. Moreover, the most significant side effects associated with octreotide can be attributed to interaction with and activation of SST2 (30). It is our hypothesis that a therapy specifically targeting SST5 would inhibit insulin secretion without the detrimental side effects seen with SST2 activation. In addition to having suboptimal efficacy/side effect profiles, octreotide and other somatostatin analogs used off-label in the treatment of HI are all injectable peptides that require subcutaneous (octreotide, lanreotide) or intramuscular (octreotide long-acting release) administration. Thus, a therapy that could be dosed orally would greatly increase the ease of treatment and remove the possibility of injection site pain and reactions. Crinetics Pharmaceuticals launched a medicinal chemistry campaign aimed to discover selective nonpeptide SST5 agonists as a new approach to managing hyperinsulinemic hypoglycemia. Those efforts lead to the discovery of CRN02481 (also known as compound 28), a very specific and potent SST5 agonist with good drug-like properties (31). Here we describe the evaluation of CRN02481 on insulin secretion particularly in the context of HI. After treatment of Sur1^−/− (also known as Abcc8^−/−) mice, a model of K_ATP-HI due to the lack of expression of the Sulfonylurea Receptor 1 subunit of the K_ATP channels, with CRN02481 we observed a significant decrease of insulin secretion both in vitro during perifusion of isolated islets and in vivo, rescuing fasting hypoglycemia. Importantly, CRN02481 treatment resulted in a significant decrease in stimulated insulin secretion in both healthy and control human islets and in pancreatic islets isolated from three different patients with HI. Taken together, these results illustrate the potential of SST5-specific therapies like CRN02481 for the treatment of HI.

Results

Selectivity of the nonpeptide SST5 agonist CRN02481 for human and mouse SST5 receptor

Chinese Hamster Ovary (CHO) cells expressing each of the human or mouse SST subtypes receptors were treated with NKH477 to increase the production of intracellular cAMP. Upon SSTR activation by the specific agonist (SS14, Octreotide, Lanreotide, Pasireotide or CRN02481), cAMP levels decreased in a concentration-dependent manner and potencies were calculated and expressed as EC₅₀s (Table 1). As expected, the native ligand SS14 was non-selective at the five human somatostatin receptors while octreotide exhibits considerable potency at hSST2 (EC₅₀ = 0.06 nM), but exhibiting less potency for hSST5 (EC₅₀ = 2.7 nM) and hSST3 (EC₅₀ = 5.2 nM). Lanreotide shows weaker activity at hSST5 (EC₅₀ = 16 nM) and hSST3 (EC₅₀ = 200 nM) making it more specific at hSST2 (EC₅₀ = 0.1 nM) than octreotide. By comparison, pasireotide is an extremely potent SST5 (EC₅₀ = 0.081 nM) agonist that still retains subnanomolar activity at both SST2 (EC₅₀ = 0.59 nM) and SST3 (EC₅₀ = 0.78 nM) (Table 1).

Table 1.

Potency of somatostatin, octreotide, lanreotide, pasireotide and CRN02481 at human and mouse somatostatin receptors

Receptor	SS14				Octreotide				Lanreotide				Pasireotide				CRN02481
	Human		Mouse		Human		Mouse		Human		Mouse		Human		Mouse		Human		Mouse
	pEC50 ± SEM	EC50 (nM)	pEC50 ± SEM	EC50 (nM)	pEC50 ± SEM	EC50 (nM)	pEC50 ± SEM	EC50 (nM)	pEC50 ± SEM	EC50 (nM)	pEC50 ± SEM	EC50 (nM)	pEC50 ± SEM	EC50 (nM)	pEC50 ± SEM	EC50 (nM)	pEC50 ± SEM	EC50 (nM)	pEC50 ± SEM	EC50 (nM)
SST1	9.0 ± 0.1	1.0	ND	ND	<5.0	>10,000	ND	ND	<5.0	>10,000	ND	ND	6.7 ± 0.2	180	ND	ND	<6.0	>1000	ND	ND
SST2	9.9 ± 0.1	0.13	10 ± 0.5	0.03	10 ± 0.1	0.06	11 ± 0.0	0.02	10 ± 0.0	0.1	10.6	0.03	9.2 ± 0.1	0.59	9.1 ± 0.1	0.88	6.4 ± 0.0	440	6.5 ± 0.1	320
SST3	9.8 ± 0.0	0.16	9.8 ± 0.1	0.15	8.3 ± 0.1	5.2	ND	ND	6.7 ± 0.1	200	ND	ND	9.1 ± 0.1	0.78	ND	ND	7.4 ± 0.1	39	8.0 ± 0.1	10
SST4	9.7 ± 0.0	0.20	ND	ND	6.4 ± 0.1	450	ND	ND	6.1 ± 0.1	820	ND	ND	<5.1	>8100	ND	ND	8.2 ± 0.1	5.7	ND	ND
SST5	10 ± 0.1	0.06	10 ± 0.1	0.07	8.6 ± 0.1	2.7	12 ± 0.1	0.002	7.8 ± 0.1	16	ND	ND	10.1 ± 0.1	0.081	10.5	0.032	9.4 ± 0.1	0.37	10 ± 0.1	0.04

ND: Not determined. pEC₅₀ and SEM given with two significant figures.

In contrast to SS14 peptide analogs, CRN02481 was designed to be very potent and selective for the human SST5 receptor (31). It shows 15-, 100-, 1200-, and >2700-fold less activity at the SST4, SST3, SST2, and SST1 receptors, respectively (Fig. 1 and Table 1 and (31)). CRN02481 exhibits a similar selectivity profile at the murine SST receptors, activating the mouse SST5 receptor with high potency (EC₅₀ = 0.041 nM) and possessing decreased activity at mouse SST2 (EC₅₀ = 320 nM) and SST3 receptors (EC₅₀ = 10 nM).

Figure 1.

Selectivity of the nonpeptide somatostatin receptor 5 (SST5) agonist CRN02481 for human SST5 receptor. Inhibition of stimulated cAMP production by CRN02481 action at the human SST receptors was evaluated in CHO-K1 cells expressing each of the human SST receptors. Concentration–response curves shown are from individual representative experiments. All points represent the mean ± SD of either triplicate or quadruplicate readings.

In addition to its selectivity within the SST receptor family, CRN02481 also showed little cross-reactivity over a panel of 64 human GPCRs. Even at a concentration of 10 μM, inhibition >70% was only observed at two receptors (dopamine transporter and adrenergic beta 2) (Table S1).

CRN02481 decreases insulin secretion from primary mouse-isolated islets

In static batch incubation of pancreatic islets isolated from wild-type (WT) mice and the hyperinsulinism mouse model Sur1^−/− mice CRN02481 significantly decreased basal levels of insulin secretion (3 mM glucose) compared to vehicle control (Fig. 2, A and B).

Figure 2.

CRN02481 decreases insulin secretion from primary isolated islets. Secreted insulin from batch incubation of (A) WT or (B) Sur1^−/− primary isolated pancreatic islets stimulated with denoted glucose concentrations and treated with either vehicle control or CRN02481 (500 nM). For WT, CRN02481 treatment is compared to vehicle control: 3 mM glucose (F(1, 42) = 8.57, p = 0.005); 10 mM glucose (F(1, 38) = 4.53, p = 0.04); 25 mM glucose (F(1, 46) = 4.68, p = 0.04). For Sur1^−/−, CRN02481 is compared to vehicle control: 3 mM glucose (F(1, 29) = 25.16, p = 2.42 × 10⁻⁵); 10 mM glucose (F(1, 34) = 19.45, p = 0.002); 25 mM glucose (F(1, 38) = 1.09, p = 0.30). Secreted insulin from batch incubation of (C) WT or (D) Sur1^−/− primary isolated islets stimulated with denoted physiological amino acid mixture (AAM) concentrations and treated with either vehicle control or CRN02481 (500 nM). For WT, CRN02481 treatment is compared to Vehicle control: 0 mM AAM (F(1, 38) = 1.39, p = 0.24); 2 mM AAM (F(1, 34) = 0.03, p = 0.86); 4 mM AAM (F(1, 38) = 0.30, p = 0.58); 10 mM AAM (F(1, 34) = 0.98, p = 0.33). For Sur1^−/−, CRN02481 is compared to Vehicle control: 0 mM AAM (F(1, 38) = 7.16, p =0.01); 2 mM AAM (F(1,38) = 15.63, p = 0.0003); 4 mM AAM (F(1, 38) = 2.96, p = 0.09), 10 mM AAM (F(1, 38) = 0.63, p = 0.43). p-values calculated with a one-way ANOVA Test. (Statistically significant comparison to Vehicle control: ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001). Data represent mean ± SD compared to vehicle control. n = 4 to 5. WT, wild type.

As we have previously described (32), Sur1^−/− islets only minimally increase insulin secretion in response to high glucose (25 mM); however, they respond to stimulation with a physiological amino acid mixture (AAM) mimicking the phenotype observed in individuals with K_ATP-HI who exhibit impaired glucose-stimulated insulin secretion (33) and protein-induced hypoglycemia (34, 35). As expected, WT islets did not secrete insulin in response to AAM, while Sur1^−/− islets demonstrated significantly increased insulin secretion in response to AAM (Fig. 2, C and D). In WT islets, CRN02481 inhibited glucose-stimulated insulin secretion at all three glucose concentrations (3, 10, and 25 mM) (Fig. 2A). Treatment with CRN02481 significantly decreased insulin secretion both basally and in response to AAM in Sur1^−/− islets to roughly basal levels when stimulated with 2 mM AAM compared to vehicle control (Fig. 2D). These results indicate that treatment with the selective nonpeptide SST5 agonist CRN02481 significantly decreases both basal and fuel-stimulated insulin secretion in primary isolated islets from the hyperinsulinism mouse model Sur1^−/− mice.

CRN02481 significantly abrogates insulin secretion and reduced cytosolic calcium in response to secretagogues

We also assessed the effect of CRN02481 on insulin secretion kinetics through the perifusion of isolated islets. Treatment with CRN02481 significantly reduced insulin secretion in WT islets in response to a glucose ramp (0–25 mM) compared to vehicle control (2-way ANOVA, p < 0.0001) (Fig. 3A). Similarly, CRN02481 treatment of Sur1^−/− islets resulted in significantly reduced insulin secretion to nearly basal levels in response to an AAM ramp (0–12 mM) compared to vehicle control (2-way ANOVA, p < 0.0001) (Fig. 3B). To further evaluate the effect of CRN02481 on insulin secretion we measured cytosolic calcium, [Ca²⁺]_c, of islets isolated from Sur1^−/− mice during perifusion. The increases in [Ca²⁺]_c in response to glucose were completely abrogated in the WT islets with the treatment of CRN02481 (Fig. 3, C and D). Likewise, in response to AAM, the flux of [Ca²⁺]_c was also decreased in Sur1^−/− islets (Fig. 3, E and F). These data demonstrate a notable suppression of insulin secretion in both WT and Sur1^−/− primary isolated islets in vitro in response to treatment with CRN02481.

Figure 3.

CRN02481 significantly abrogates insulin secretion and calcium flux in response to glucose and amino acid stimulus. Perifusion of primary isolated islets to assess insulin release in (A) WT islets stimulated with glucose ramp (0–25 mM) and KCl (30 mM) or (B) Sur1^−/− islets stimulated with amino acid (AA) ramp (0–12 mM) and KCl (30 mM). Two-way ANOVA with Tukey’s multiple comparisons revealed that insulin release is significantly decreased with CRN02481 (500 nM) treatment for both WT (F(1, 384) = 94.75, p < 0.0001) and Sur1^−/− (F(1,384) = 221.2, p < 0.0001) islets when compared to vehicle. (n = 3). Intracellular Ca²⁺ measurement of primary isolated islets for assessment of calcium signaling in WT islets treated with glucose steps (3 mM, 9 mM, 27 mM), KCl (30 mM), and (C) vehicle or (D) 500 nM CRN02481. Sur1^−/− islets treated with amino acid steps (4 mM, 8 mM, and 12 mM), KCl (30 mM), and (E) vehicle or (F) 500 nM CRN02481. Two-way ANOVA with Tukey’s multiple comparisons demonstrated that treatment with CRN02481 resulted in significantly decreased intracellular Ca²⁺ signaling in both WT (F(1, 3660) = 56.60, p < 0.0001) and Sur1^−/− islets (F(1, 3660) = 799.0, p < 0.0001) compared to vehicle (n = 3–6). Data represent mean ± SD compared. WT, wild type.

Treatment with CRN02481 increases plasma glucose and decreases insulin secretion in both WT and Sur1^−/− mice

With the effect of CRN02481 on insulin secretion evident in isolated islets, we assessed the response in vivo. Fasting plasma glucose significantly increased at 1 and 2 h after dosing with CRN02481 in WT mice compared to vehicle-treated mice (Fig. 4A). Although absolute plasma insulin levels did not show a significant decrease (Fig. 4B), the ratio of insulin to glucose at 1 and 2 h was significantly decreased compared to control (Fig. 4C). Fasting plasma β-hydroxybutyrate concentration was also significantly increased at 2 h after CRN02481 treatment compared to controls in WT mice (Fig. 4D), supportive of insulin suppression. Interestingly, CRN02481 treatment of Sur1^−/− mice demonstrated effects similar to that seen in WT mice. In Sur1^−/− mice we observed a significant increase in fasting plasma glucose concentration, with no significant decrease in absolute plasma insulin concentration and a significant decrease in the insulin-to-glucose ratio (Fig. 4, E–G). Sur1^−/− mice also demonstrated a significant increase in fasting plasma β-hydroxybutyrate at 1 and 2 h after CRN02481 treatment compared to vehicle-treated mice (Fig. 4H).

Figure 4.

Treatment with the SST5 agonist, CRN02481, after fasting increases plasma glucose and decreases insulin secretion in both WT and Sur1^−/−K_ATP-HI. Fasting evaluation after overnight fast (16 h) at baseline (0 h), 1 h, or 2 h post-treatment with either vehicle or CRN02481 (30 mg/kg) of WT mice assessing (A) plasma glucose [Baseline: (F(1, 12) = 38.46, p = 4.57 × 10^-5); 1 h (F(1, 12) = 8.04, p = 0.02); 2 h (F(1, 12) = 12.11, p = 0.005)], (B) plasma insulin [Baseline (F(1, 12) = 0.0004, p = 0.98); 1 h (F(1, 12) = 3.14, p = 0.10; 2 h (F(1, 12) = 2.76, p = 0.12)], (C) insulin/glucose ratio [Baseline (F(1, 12) = 0.01, p = 0.91); 1 h (F(1, 12) = 4.50, p = 0.05); 2 h (F(1, 12) = 14.79, p = 0.002)], and (D) β-hydroxy butyrate [Baseline (F(1, 12) = 3.08, p = 0.10); 1 h (F(1, 12) = 3.818, p = 0.07); 2 h (F(1, 12) = 33.64, p = 8.47 × 10^-5)]. Fasting evaluation after overnight fast (16 h) at baseline (0 h), 1 h, or 2 h post-treatment with either vehicle or CRN02481 (30 mg/kg) of Sur1^−/− mice assessing (E) plasma glucose [Baseline (F(1, 12) = 0.18, p = 0.67; 1 h (F(1, 12) = 25.87, p = 0.0002); 2 h (F(1, 12) = 42.59, p = 2.83 × 10^-5)], (F) plasma insulin [Baseline (F(1, 12) = 0.40, p = 0.54); 1 h (F(1, 12) = 1.09, p = 0.31); 2 h (F(1, 12) = 1.67, p = 0.221)], (G) insulin/glucose ratio [Baseline (F(1, 12) = 0.54, p = 0.48); 1 h (F(1, 12) = 8.36, p = 0.01); 2 h (F(1, 12) = 6.94, p = 0.02)], and (H) β-hydroxybutyrate [Baseline (F(1, 12) = 2.35, p = 0.15); 1 h (F(1, 12) = 12.25, p = 0.004); 2 h (F(1, 12) = 21.23, p = 0.0006)]. (n = 7) p-values calculated with one-way ANOVA Test compared to vehicle control (∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05). Data represent mean ± SD.

Upon challenge with a glucose tolerance test (IPGTT), we observed significantly increased plasma glucose and decreased plasma insulin levels in WT mice after treatment with CRN02481 compared to vehicle-treated mice (Fig. 5, A–C). Similarly, a significant increase in plasma glucose and a significant decrease in plasma insulin was observed in Sur1^−/− mice after glucose challenge compared to vehicle-treated mice (Fig. 5, D–F). These results demonstrate that treatment with CRN02481 significantly decreases insulin secretion and abrogates fasting hypoglycemia in the Sur1^−/− hyperinsulinism mouse model.

Figure 5.

SST5 agonist, CRN02481, increases plasma glucose and decreases insulin secretion in both WT and Sur1^−/−K_ATP-HI mouse models after glucose challenge.A, glucose tolerance test (GTT) in WT mice treated with vehicle or CRN02481 (30 mg/kg) by gavage followed by I.P. injection of 2 g/kg glucose. Two-way ANOVA with Tukey’s multiple comparisons: (F(1, 60) = 80.45, p < 0.0001). (n = 7) (B) Area under the curve (AUC) calculation for GTT of WT mice. One way ANOVA: (F(1, 12) = 15.98, p = 0.002). C, plasma insulin levels of WT mice during 0, 30, and 60 min time points during GTT. One-way ANOVA: 0 min (F(1, 12) = 0.005, p = 0.94); 30 min (F(1, 12) = 30.88, p = 0.0001); 60 min (F(1, 12) = 12.43, p = 0.004). D, GTT in Sur1^−/− mice treated with vehicle or CRN02481 (30 mg/kg) by gavage followed by I.P. injection of 2 g/kg glucose. Two-way ANOVA with Tukey’s multiple comparisons: (F(1, 60) = 124.3, p <0.0001) (n = 7). E, AUC calculated for Sur1^−/− mice GTT. One-way ANOVA: (F(1, 12) = 56.67, p = 6.97 × 10^-6). F, plasma insulin levels of Sur1^−/− mice during 0, 30, and 60 min time points during GTT. One-way ANOVA: 0 min (F(1, 12) = 0.105, p = 0.75); 30 min (F(1, 12) = 13.16, p = 0.003); 60 min (F(1, 12) = 6.67, p = 0.02). (n = 7) (Comparison to vehicle control: ∗∗∗p < 0.001; ∗∗p <0.01; p < 0.05). Data represent mean ± SD. WT, wild type.

Effect of somatostatin, somatostatin peptide analogs, and diazoxide on glucose- and tolbutamide-stimulated insulin secretion in isolated healthy human islets

To have a better comparison between a specific SST5 nonpeptide like CRN02481, somatostatin, SSAs, and diazoxide, we evaluated the effect of these molecules in glucose- and tolbutamide-stimulated insulin secretion in healthy human islets from several donors (Table 2). Tolbutamide is a sulfonylurea that blocks the K_ATP channel resulting in insulin secretion while diazoxide is a K_ATP channel opener that causes the opposing effect and suppresses insulin secretion. As expected, SS14 and analogs (octreotide, lanreotide, and pasireotide) suppressed insulin secretion in human islets stimulated with high glucose (16.7 mM) alone or high glucose (16.7 mM) with tolbutamide (0.1 μM). SS14 produced the strongest effect, suppressing ∼80% insulin in both conditions. The peptide analogs had similar effects among them, suppressing insulin ∼65 to 70% (Fig. 6, A and B). These results confirm the few reports demonstrating the inhibitory effect of SS14 in static assays in human islets (28, 36) and octreotide in perfused human pancreas (29). These data also show the activity of lanreotide and pasireotide in the dynamic secretion of insulin in isolated human islets for the first time. Because the three analogs each possess mixed pharmacology at the SST family of receptors (Table 1), it is difficult to deconvolute the contribution of each receptor to the anti-secretory activity observed. However, we have shown that specific and potent small molecules targeting SST3 and SST4 receptors (EC₅₀ = < 1 nM) have no effect on glucose- and tolbutamide-stimulated insulin secretion in healthy human islets (37). Copy of the poster can be found here: https://crinetics.com/pipeline/crn04777-oral-sst5-agonist-congenital-hyperinsulinism-congenital-hi/). Therefore the anti-secretory activity of these peptide agonists is due to their activation of SST2 and SST5 receptors. The ability of SS14 and analogs to suppress tolbutamide-stimulated insulin secretion is also shown here for the first time and confirms these peptides act downstream of the closure of the K_ATP channel. Diazoxide suppressed glucose-stimulated insulin secretion at concentrations 1000-fold higher than SS14 and importantly, did not suppress tolbutamide-stimulated insulin secretion (Fig. 6, C and D), mimicking its inability to suppress insulin secretion in children with K_ATP- HI.

Table 2.

Donor information

Prodo ID	Gender	Ethnicity	Age (y)	BMI	HbA1C (%)	Causeofdeath	Islet index	Islet purityby count (%)	Isletviability	Islet sterilitygram stain	Isletsused infigure
HP-20066-01	Female	Caucasian	60	25.8	5.1	Stroke	0.64	90	95	Negative	6D
HP-20191-01	Female	Caucasian	39	22.1	5.6	Head trauma	0.70	95	95	Negative	6C and 6D
HP-20262-01	Male	Asian	67	24.4	5.8	Stroke	0.54	90–95	95	Negative	6B
HP-20281-01	Male	Caucasian	55	25.6	4.9	Stroke	1.14	90	95	Negative	6B
HP-20317-01	Male	Hispanic	58	26.8	4.9	Stroke	0.67	90	95	Negative	6B
HP-20341-01	Male	Hispanic	35	18.1	5.3	Stabbing	1.00	90	95	Negative	6B
HP-21100-01	Male	African American	66	34.3	5.5	Head trauma	0.85	90	95	Negative	6D
HP-21141-01	Male	Caucasian	55	21.3	5.6	Head trauma	0.65	90	95	Negative	6A, 6B, 6D and 7B
HP-21155-01	Male	Hispanic	18	20.4	5.4	Head trauma	1.00	85–90	95	Negative	6D and 7B
HP-21167-01	Male	Caucasian	46	28.1	5.0	Stroke	0.83	85	95	Negative	6D, 7A and 7B
HP-21203-01	Male	Hispanic	25	26.5	5.8	Head trauma	1.50	95	95	Negative	7B
HP-21189-01	Male	Hispanic	36	26.3	5.4	Head trauma	1.10	90	95	Negative	6B

Figure 6.

Suppressive effect of SS14, peptide analogs and diazoxide on insulin secretion in islets from healthy donors. Isolated healthy human islets (Prodo Labs, CA) were loaded in a perifusion system (Biorep, FL) and treated over time with 3 mM glucose (G3), 16.7 mM glucose (G16.7), and 16.7 mM glucose + 100 μM tolbutamide (G16.7 + Tolb) in the presence or absence of SS14 and analog peptides, pasireotide, octreotide and lanreotide at 0.1 μM (A and B) and diazoxide at 10 μM and 100 μM (C and D). Insulin secretion was quantified using an ELISA assay (Mercodia, Uppsala, Sweden) and the dynamic stimulation index (dSI) was calculated as stimulated insulin levels/basal insulin levels. (A and C) Mean dSI ± SD (n = 2 technical replicates) from one representative donor. (B and D) Percent insulin secretion was calculated in each condition and compared to the vehicle group which was set to 100%. One-way ANOVA analysis with Dunnett’s multiple comparisons of peptide (B): G16.7 (F(4, 16) = 38.76, p < 0.0001 for all peptide comparisons to vehicle) and G16.7 + Tolb (F(4, 15) = 24.32, p < 0.0001 for all comparisons to vehicle); diazoxide (D): G16.7 (F(2, 12) = 45.27, p = 0.25 for 10 μM, p < 0.0001 for 100 μM compared to vehicle) and G16.7 + Tolb (F(2, 12) = 0.039, p = 0.99 for 10 μM and p = 0.94 for 100 μM compared to vehicle). Data represent mean ± SD from 4 to 6 independent donors in (B) and 3 to 6 independent donors in (D). (Comparison to Vehicle control: ∗∗∗∗, p ≤ 0.0001).

Insulin secretion in isolated pancreatic islets from healthy human donors and human patients with HI is significantly suppressed by treatment with CRN02481

With significant downregulation of insulin secretion evident in the mouse models, and the effect of the peptides and diazoxide as controls and comparators, we evaluated the effect of CRN02481 on glucose- and tolbutamide-stimulated insulin secretion in isolated human islets from four donors (Table 2). Perifusion with either vehicle or increasing concentrations of CRN02481 (10 nM, 100 nM, or 1000 nM) was performed on isolated healthy, control human islets accompanied by stimulation with high glucose alone or high glucose with tolbutamide. CRN02481 significantly reduced both high glucose- and tolbutamide-stimulated insulin secretion in a concentration-dependent manner with the maximum effect at 1000 nM (Fig. 7, A and B). These results indicate that activation of SST5 by CRN02481 resulted in the expected anti-secretory effect on insulin in healthy human islets and more importantly showed that the compound worked downstream of the K_ATP channel having the ability to also suppress tolbutamide-stimulated insulin secretion, in contrast to diazoxide (Fig. 6, C and D). We were also able to assess the effect of CRN02481 in islets from patients with HI. Perifusion was performed on pancreatic islets isolated from tissue collected after the pancreatectomy of three patients with HI (Table 3). The first patient had hyperinsulinism associated with Beckwith-Wiedemann Syndrome due to uniparental paternal isodisomy for chromosome region 11p(UPD-BWS) (38). Distinct from K_ATP-HI, BWS-HI patients exhibit an exaggerated insulin secretion in response to glucose leading to hypoglycemia (39). Treatment with CRN02481 significantly decreased insulin secretion during perifusion with a glucose ramp (3–25 mM) compared to vehicle control (Fig. 7, C and D). Genetic testing of the second patient revealed two heterozygous recessive mutations in ABCC8, which resulted in diazoxide-unresponsive diffuse K_ATP-HI. Similar to islets from Sur1^−/− mice, K_ATP-HI islets only minimally increase insulin secretion in response to high glucose (25 mM) but do respond to stimulation with a physiological amino acid mixture (33, 34). Perifusion of these pancreatic islets exhibited a significant decrease in basal insulin secretion (min 20–30) and in response to an AAM ramp (0–12 mM) with CRN02481 treatment compared to vehicle control (Fig. 7, E and F). The third patient was diagnosed with diffuse K_ATP-HI caused by a mutation of ABCC8 (c.3577delG) and deletion of ABCC8 exon 20 and 29. Similar to the second patient, perifusion results showed that CRN02481 significantly decreased basal insulin secretion (min 20–30) and in response to the AAM ramp compared to vehicle control (Fig. 7, G and H). Further analysis revealed no change in glucagon secretion with the treatment of CRN02481 in islets from these three HI patients (Fig. S1). Remarkably, CRN02481 treatment of human islets from patients with hyperinsulinism demonstrated a significant decrease in insulin secretion and illustrates the feasibility of SST5-specific therapies for the treatment of HI.

Figure 7.

CRN02481 significantly curtails insulin secretion in pancreatic islets from human healthy donors and patients with HI. Isolated human healthy, control islets (Prodo Labs, CA) were loaded in a perifusion system (Biorep, FL) and treated over time with 3 mM glucose (G3), 16.7 mM glucose (G16.7), and 16.7 mM glucose + 100 μM tolbutamide (G16.7 + Tolb) in the presence of absence of increasing concentrations of CRN02481 as described in material and methods. Insulin secretion was quantified using an ELISA assay (Mercodia, Uppsala, Sweden) and the dynamic stimulation index (dSI) was calculated as stimulated insulin levels/basal insulin levels. (A) Mean dSI ± SD (n = 2 technical replicates) from one representative donor. B, percent insulin secretion was calculated in each condition and compared to the vehicle group which was set to 100%. One-way ANOVA analysis with Dunnett’s multiple comparisons: G16.7 (F(3, 12) = 10.21, p = 0.98 for 0.01 μM, p = 0.04 for 0.1 μM, and p = 0.002 for 1 μM compared to vehicle); G16.7 + Tolb (F(3, 12) = 14.49, p = 0.77 for 0.01 μM, p = 0.01 for 0.1 μM, and p = 0.001 for 1 μM compared to vehicle). Data represent mean ± SD from 4 independent donors compared to vehicle. C, Perifusion of primary isolated BWS-HI human islets to assess insulin release with treatment of CRN02481 (500 nM) in response to glucose ramp (0–25 mM) and KCl (Two-way ANOVA with Tukey’s: (F(1, 95) = 146.3, p < 0.0001). D, percent insulin release for BWS-HI (Patient #1) human islet perifusion. E, Perifusion of primary isolated K_ATP-HI human islets (Patient #2) to assess insulin release with treatment of CRN02481 (500 nM) in response to AAM ramp (0–12 mM) and KCl (Two-way ANOVA with Tukey’s: (F(1, 95) = 119.4, p < 0.0001). F, percent insulin release for K_ATP-HI Patient #2 perifusion. G, Perifusion of primary isolated K_ATP-HI human islets (Patient #3) (Two-way ANOVA with Tukey’s: (F(1, 95) = 163.5, p < 0.0001). H, percent insulin release for K_ATP-HI perifusion (Patient #3). (Compared to vehicle: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001). Data represent mean ± SD compared to vehicle.

Table 3.

Clinical characteristics of HI islets donors

Subject characteristics	HI patient #1	HI patient #2	HI patient #3
Gender	Male	Male	Male
Ethnicity	Caucasian	Black	Caucasian
Age (y)	0.18	0.16	0.1
Weight-for-length	68 percentile	82 percentile	78 percentile
HbA1C	4.8	4.7	ND

Discussion

The only FDA-approved treatment for congenital hyperinsulinism is diazoxide, a potent K_ATP channel opener. Unfortunately, diazoxide is ineffective for the treatment of the most common and severe form of hyperinsulinism, K_ATP-HI. Therefore, the discovery of a therapy that bypasses the canonical K_ATP channel mechanism is critical for the treatment of K_ATP-HI and other diazoxide-unresponsive cases, as well as represents a potential alternative to diazoxide treatment. The pulsatile synchronicity shared between insulin and somatostatin presents a unique regulatory relationship and presents an opportunity to harness the inhibitory regulation of somatostatin on insulin secretion. Importantly, somatostatin receptor signaling effects occur late in the insulin secretion pathway and are downstream of known HI-causing mutations, therefore a potent somatostatin agonist should be effective against all forms of HI.

Previous studies have identified the ability of somatostatin to decrease the amplitude of insulin pulses while having no effect on the pulse cycles (40, 41). Additionally, somatostatin abrogates insulin secretion by binding to and activating SST receptors and inhibitory signaling action on adenylate cyclase followed by a decrease in cAMP or activation of calcineurin (42, 43, 44). Therefore, somatostatin is an attractive potential therapy for HI due to its insulin secretion inhibitory abilities involving a mechanism distinct from the K_ATP channel. Unfortunately, the use of SS14 is severely hampered by a short half-life in circulation. To counter the limiting short half-life of endogenous somatostatin, stabilized somatostatin peptide analogs such as octreotide and lanreotide were generated. These peptides primarily target SST2 with subnanomolar activity, but still have additional potency at the SST5 and SST3 receptors (Table 1). Both octreotide and lanreotide are limited by potentially harmful side effects linked to potent SST2 affinity, such as glucagon suppression and the observation of necrotizing enterocolitis, as well as a requirement for parenteral administration (6, 7). Pasireotide is a second-generation synthetic polypeptide analog of somatostatin, which boasts a 40-fold higher affinity for SST5 than octreotide but maintains a comparable affinity for SST2 and also targets the SST3 receptor (Table 1 and (45)). Interestingly, pasireotide causes less inhibition of glucagon secretion through SST2 than first-generation somatostatin analogs but does not completely prevent it (46). A single case report describes the successful use of pasireotide to control persistent hypoglycemia of an adult with hyperinsulinemic hypoglycemia (47). However, a more recent case report using pasireotide for the treatment of a severe K_ATP-HI case indicates that while no side effects were observed, both short- and long-acting pasireotide injections were insufficient to maintain glycemic control (48).

In this study, pure lines of WT (C57BL/6J) and Sur1^−/− male mice were used for experiments. While we acknowledge these limitations, it is important to note that in vitro and in vivo data in this study including perifusion, fasting plasma glucose, plasma insulin, glucose tolerance, and insulin-to-glucose ratios, were comparable to that we have seen previously using littermate controls (32, 49). The Sur1^−/− mouse model of K_ATP-HI exhibits a fasting hypoglycemia phenotype similar to the human phenotype (50), moreover, the islet phenotype demonstrates the same characteristics as human islets isolated from infants with hyperinsulinism due to inactivating K_ATP channel mutations: high cytosolic calcium concentration and altered fuel-stimulated insulin secretion with lack of response to stimulation with glucose and increased amino-acid stimulated insulin secretion (32, 51). The altered fuel-stimulated insulin secretion explains the in vivo phenotype of impaired glucose tolerance and protein-induced hypoglycemia (33, 34). Importantly, we have used the Sur1^−/− mouse model for proof-of-concept studies for the development of other therapeutic approaches (49, 52, 53), which have been successfully translated to studies in affected patients (54, 55). Only male mice were used for this study, but we do not expect to see a different response in female Sur1^−/− mice since they also exhibit the same phenotype as male mice (fasting hypoglycemia, glucose intolerance). An SST5 agonist that has moved forward into clinical studies has addressed this limitation (56). Sur1^−/− mice and WT mice received CRN02481 orally at a dose of 30 mg/kg based on a short-term dose-finding study where the effect of different doses on fasting glucose was evaluated (57).

Selective nonpeptide agonists of somatostatin receptors are the next evolution of somatostatin-related treatment for HI, which allows for more specific SST receptor selectivity and, importantly, the ability to be dosed orally. Our characterization of CRN02481, an SST5 selective nonpeptide agonist, discovered after a medicinal chemistry campaign aimed to find SST5 agonists (31), has clearly demonstrated its ability to significantly decrease insulin secretion similar to SS14 and its analogs and to be more potent and effective than diazoxide in suppressing tolbutamide-stimulated insulin secretion in healthy human islets. CRN02481 also prevents fasting hypoglycemia in an in vivo model of K_ATP-HI and more importantly it exhibited anti-secretory effects on insulin in islets from HI patients. The high selectivity of CRN02481 for SST5 reduces the likelihood of having unspecific actions or an effect on glucagon secretion and other potential side effects of SST2 activation. Our own unpublished data indicates that another very specific SST5 agonist, similar to CRN02481 and equally potent, does not have any effect on the dynamic secretion of glucagon in response to arginine in isolated healthy human islets from eight independent donors. These results show the clear potential of SST5 selective agonists to be developed as a future therapy for patients with diazoxide unresponsive K_ATP-HI as well as have broad potential for other patients with HI.

Experimental procedures

CRN02481 compound details

CRN02481, 3-{4-[(3S)-3-aminopyrrolidin-1-yl]-5-(4-methyl-1H-1,3-benzodiazol-2-yl)pyridin-3-yl-5-fluorobenzonitrile was discovered, synthesized, and characterized as described in detail in (31). It possesses good stability when incubated with both human or rat liver microsome preparations, and exhibits good oral bioavailability in preclinical species, exhibiting a 32% oral bioavailability in rats and a 55% oral bioavailability in dogs (58).

Inhibition of stimulated cAMP production by CRN02481 action at the human and mouse SST receptors

SST subtypes are G_i/o-coupled GPCRs that inhibit adenylyl cyclase (AC) activity, resulting in a decrease of intracellular cyclic AMP (cAMP) upon activation by the agonist. We used homogeneous time-resolved fluorescence technology (HRTF cAMP kit, Cisbio) to evaluate agonist potency at various SSTR subtypes by measuring intracellular cAMP concentrations.

CHO cells stably expressing one of the human or mouse SSTR subtypes were grown and treated as described in detail in Zhao et al (31). In brief, cells cultured for 4 days in a 96-well plate format were treated with NKH477, a soluble forskolin analog that induces the production of cAMP, plus several concentrations of SS14, Octreotide, Lanreotide, Pasireotide or CRN02481, in assay buffer for 20 min at 37 °C. The final concentration of agonists ranges from 0 to 10000 nM. cAMP levels were measured after lysis following the instructions provided by Cisbio. The time-resolved fluorescent signal was read in a multi-plate reader and cAMP concentrations calculated by regression to a standard curve. Agonists potency (EC₅₀) was determined by plotting agonist concentration vs cAMP levels and using standard curve-fitting methods. All data manipulations were performed using GraphPad Prism v6 or v7 (GraphPad, San Diego. CA)

Animal studies

WT and Sur1^−/− mice studies were conducted at the Children’s Hospital of Philadelphia and approved by the Institutional Animal Care and Use Committee (IACUC).

The generation and genotyping of Sur1^−/− mice were previously described (59). Mice are maintained in our mouse colony in a C57Bl/6 genetic background. Male, 8 to 10 weeks old, Sur1^−/− mice were generated via Sur1^−/− male x Sur1^−/− female crosses and used in all experiments. Mice were maintained on a 12:12-h light-dark cycle and were fed a standard rodent chow diet (LabDiet, Picolab rodent diet 5053).

WT C57BL/6J male mice were acquired from Jackson laboratories, (Bar Harbor, ME) at 8 to 10 weeks of age. Mice were allowed to acclimate to a 12:12-h light-dark cycle, housing humidity and temperature for at least 72 h prior to initiation of the study. Mice were group-housed and maintained on a standard rodent diet (LabDiet, Picolab rodent diet 5053). All animals were provided free access to drinking water.

Fasting evaluation

Sur1^−/− mice and WT mice received CRN02481 orally at a dose of 30 mg/kg or an equal volume of vehicle (25 mM Citrate Buffer, pH 3) after an overnight fast (16–18 h). This dose was selected based on a short-term dose-finding study where the effect of different doses on fasting glucose was evaluated (57). A copy of the poster can be found here: https://crinetics.com/pipeline/crn04777-oral-sst5-agonist-congenital-hyperinsulinism-congenital-hi/). Plasma glucose and β-hydroxybutyrate levels (BHB) were assessed at baseline (0 h), 1, and 2 h after treatment by a portable glucose meter (Stat Strip Xpress Glu, Nova biomedical) and Ketone meter (Nova Max plus, Nova biomedical). Blood was obtained from a tail nick. Blood was collected for measurement of plasma insulin levels using Mouse Ultrasensitive Insulin ELISA kit (Catalog # 80-Insmsu-E10, Alpco, RRID:AB_2792981).

Glucose tolerance test

Sur1^−/− mice and WT mice received CRN02481 orally at a dose of 30 mg/Kg or an equal volume of vehicle (25 mM Citrate Buffer, pH 3) after an overnight fast (16 h). One hour after administration of CRN02481 or vehicle, dextrose (2 g/kg) was given by i.p. injection. Plasma glucose concentrations were measured at baseline and every 30 min for 2 h. Blood was also obtained at baseline (0 h), 30 min, and 60 min for measurement of plasma insulin by ELISA (Catalog # 80-Insmsu-E10, Alpco).

Pancreatic islet batch incubations, perifusions, and intracellular calcium imaging

Human HI islets were isolated from fresh pancreata from surgical specimens from neonates procured through an institutional review board–approved protocol at the Congenital Hyperinsulinism Center at the Children’s Hospital of Philadelphia (Table 3). The pancreas was injected with collagenase (Sigma-Aldrich) and digested. Islets were handpicked under microscopy and cultured in RPMI-1640 medium containing 5 mM glucose for 2 to 3 days prior to perifusion studies. Sur1^−/− and WT mouse (8–16 weeks old) islets were digested (Collagenase P, Roche), isolated, and then cultured for 72 h in RPMI 1640 (GIBCO 11879) medium supplemented with 10 mM glucose, 10% fetal bovine serum, 10% glutamine, and 10% penicillin/streptomycin at 37 °C in a 5% CO2, 95% air-humidified incubator.

For static incubations, 4 to 5 replicates with 5 islets each were treated with CRN02481 (100 nM) or vehicle (DMSO) and glucose or physiological amino acid mix (AAM) for 1.5 h and then the supernatant was collected to assess secreted insulin by Homogeneous Time Resolved Fluorescence (HTRF) (Catalog # 62IN1PEH, CisBio, RRID:AB2890910). The physiological mixture of 20 amino acids was previously described (51). For perifusion, 300 cultured (mouse or human) islets were loaded onto a nylon filter in a chamber and perifused with Krebs-Ringer bicarbonate buffer (KRBB) (115 mmol/liter NaCl, 24 mmol/liter NaHCO3, 5 mmol/liter KCl, 1 mmol/liter MgCl2, 2.5 mmol/liter CaCl2, 10 mm HEPES, pH 7.4) with 0.25% bovine serum albumin at a flow rate of 1 ml/min. During perifusion islets were treated with vehicle (DMSO) or CRN02481 (500 nM) or vehicle at time 0 and then stimulated with a glucose ramp (3–25 mM) or AAM ramp (0–12 mM) for 40 min followed by a 20 min wash in KRBB and finally KCl (30 mM) (20 min). Fractions were collected at a rate of 1 ml/min and were assessed for secreted insulin or glucagon (Catalog # 62CGLPEG, CisBio, RRID:AB_2936335) by HTRF. For intracellular Ca²⁺ measurement, islets were pre-incubated with the Fura 2 fluorescent probe, treated with CRN02481 (500 nM) or vehicle, and then exposed to increasing concentrations of glucose or AAM. Measurement of intracellular Ca²⁺ was calculated as the ratio of the excitation of Fura 2 at 334 and 390 nm.

Human pancreatic islets, obtained from Prodo Labs, (Aliso Viejo. CA) were cultured in PIM(S) media supplemented with PIM(ABS), PIM(3X), and PIM(G) (Prodo Labs) following the manufacturer indications. Perifusion experiments were performed using the PERI5 perifusion system (Biorep Technologies) with islets from several independent healthy donors (Table 2). Briefly, human islets were equilibrated with low glucose (3 mM) for 74 min, followed by high glucose (16.7 mM) for 21 min, and high glucose (16.7 mM) plus tolbutamide (100 μM) for another 14 min in the presence or absence of increasing concentrations of CRN02481 (10 nM, 100 nM, and 1000 nM). Perifusion ended with low glucose (3 mM) for 14 min. Fractions were collected at 4 °C at a rate of 100 μl/min and insulin was measured using a commercially available ELISA kit (Catalog # 10–1132–01, Mercodia, RRID:AB_2783838). The dynamic stimulation index was calculated by dividing the insulin concentration in response to any given stimulus at any time by the average basal insulin level which was calculated as the mean insulin levels during the last 10 min of the equilibration period. The area under the curve (AUC) during CRN02481 treatments was calculated and compared to the average AUC of the vehicle group which was set at 100%. All data manipulations were performed using GraphPad Prism v8 or v9 (GraphPad, San Diego, CA, RRID:SCR_002798).

Statistics

Statistical analyses were performed on Excel or GraphPad Prism software v8 or v9. Results are presented as mean ± standard deviation (SD). The level of significance was set at p < 0.05. For multiple measurements, data were analyzed using 2-way ANOVA Repeated Measures, Tukey’s multiple comparison test. Single time endpoints data were analyzed using a one-way ANOVA Test. Differences were considered significant at p < 0.05.

Data availability

All the data resulting from these studies are contained within the manuscript or available upon request to corresponding author: Diva D. De Leon at deleon@chop.edu.

Supporting information

This article contains supporting information.

Conflict of interest

P. A., E. R. B. and S. F. B work for Crinetic Pharmaceuticals. D. D. D. L. has received consulting fees from Crinetics Pharmaceuticals, Zealand Pharma, Hanmi Pharmaceutical, Eiger Biopharmaceuticals, and Heptares Therapeutics. D. D. D. L. has received research funding from Tiburio Therapeutics, and Twist Pharma for studies not included in this manuscript.

Reviewed by members of the JBC Editorial Board. Edited by Mike Shipston