Pyridyl-alanine as a Hydrophilic, Aromatic Element in Peptide

Aug 10, 2016 - Department of Chemistry, Indiana University, 800 East Kirkwood, Bloomington, Indiana 47405 United States. ‡ Department of Medicine ...
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Pyridyl-alanine as a Hydrophilic, Aromatic Element in Peptide Structural Optimization Piotr A. Mroz, Diego Perez-Tilve, Fa Liu, Vasily Gelfanov, Richard D. DiMarchi, and John P. Mayer J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00840 • Publication Date (Web): 10 Aug 2016 Downloaded from http://pubs.acs.org on August 21, 2016

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

Pyridyl-alanine as a Hydrophilic, Aromatic Element in Peptide Structural Optimization

Piotr A. Mroz1, Diego Perez-Tilve 2, Fa Liu 3, Vasily Gelfanov3, Richard D. DiMarchi1,3*, and John P. Mayer3*

1

Department of Chemistry, Indiana University, Bloomington, IN 47405 USA. 2Department of

Medicine, Metabolic Diseases Institute, University of Cincinnati, Cincinnati, OH, 45267 USA. 3

Novo Nordisk Research Center, Indianapolis, IN 46241.

Dedicated to the memory of William W. Bromer, Ph.D and the inspiration provided by his multiple contributions to the science of glucagon, beginning with its sequence determination.

ABSTRACT: Glucagon (Gcg) 1 serves a seminal physiological role in buffering against hypoglycemia, but its poor biophysical properties severely complicate its medicinal use. We report a series of novel glucagon analogs of enhanced aqueous solubility and stability at neutral pH, anchored by Gcg[Aib16]. Incorporation of 3- and 4-Pyridyl-alanine (3-Pal and 4-Pal) enhanced aqueous solubility of glucagon while maintaining biological properties. Relative to native hormone, analog 9 (Gcg[3-Pal6,10,13, Aib16]) demonstrated superior biophysical character, better suitability for medicinal purposes, and comparable pharmacology against

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insulin-induced hypoglycemia in rats and pigs. Our data indicate that Pal is a versatile surrogate to natural aromatic amino acids and can be employed as an alternative or supplement with isoelectric adjustment to refine the biophysical character of peptide drug candidates.

INTRODUCTION Glucagon (1) is a 29 amino acid pancreatic hormone whose primary physiological role is the mobilization of hepatic glucose through stimulation of glycogenolysis and gluconeogenesis.1 The glucagon response is an essential feature of healthy physiologic glycemic control and is often impaired in diabetes.2,

3

This deficiency presents an acute risk for insulin dependent

diabetics who regularly encounter hypoglycemia in the course of insulin therapy. In severe instances of hypoglycemia patients often require emergency intervention in the form of a subcutaneous injection of glucagon. Glucagon is supplied as a lyophilized powder which requires reconstitution in a sterile, acidic diluent immediately prior to its administration, due to the tendency of the formulated native hormone to undergo chemical degradation4, 5 and to form insoluble fibrils.6, 7 These adverse biophysical properties also complicate co-administration of glucagon with insulin via a bi-hormonal pump8,

9

and its evaluation in a number of

exploratory settings, including mini-dosing regimens.10, 11

Glucagon (1) HSQGTFTSDY SKYLDSRRAQ DFVQWLMNT

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A number of approaches have been advanced to develop a soluble formulation of native glucagon including the use of aqueous12,

13

and non-aqueous formulations.14 Independently,

DiMarchi and co-workers have introduced primary sequence modifications that reduce aggregation and chemical degradation. These involved a reduction in the number of native amide side-chains including the substitution of the acid-sensitive Gln3 with a chemically stable mimetic.15 Additionally, the substitution of Ser16 with aminoisobutyric acid (Aib) not only improved biophysical properties as reflected by minimized propensity to physical aggregation, but prevented chemical degradation via isoaspartimide formation between Asp15 and Ser16.1517

Separately from the chemical and physical stabilization of the peptide, two approaches have

been taken to enhance its aqueous solubility. These include the C-terminal extension with the exendin-derived nine amino acid sequence18 and the introduction of anionic charge to the Cterminus to decrease the isoelectric point, as a means to increase neutral pH solubility.15, 17 The so-called “aromatic patch” of glucagon comprising aromatic residues Phe6, Tyr10 and Tyr13 contributes significantly to its fibrillation tendency.19,

20

Unfortunately, this region

and particularly residues Phe6 and Tyr10 are also essential for glucagon receptor activation.17 The 3-Pal substitution has been employed primarily to improve potency, and while enhanced solubility was noted, the pyridyl side-chain was never systematically promoted for this single virtue. An early series of LHRH agonists disclosed by Folkers utilized D-3-Pal as an isostere for tryptophan, noting improvements in potency and unexpectedly, solubility.21, 22 Hirschmann and co-workers observed that replacement of a phenyl sidechain with pyridine in a series of somatostatin analogs enhanced solubility and maintained in vitro potency.23 More recently this method was successfully used to increase solubility in a series of cyclic CGRP antagonist

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peptides.24 Here we present a systematic evaluation of single and multiple 3-Pal and 4-Pal substitutions at position 6, 10, 13 and 22 of glucagon in the presence of an Aib16 substitution (Table 1 and Figure 1), aimed to optimize its aqueous solubility and stability.

Figure 1. Structures and nature of amino acid modification used in glucagon analogs [Table 1], native amino acids in black, 3-Pyridyl-alanine in red, 4-Pyridyl-alanine in green.

RESULTS The novel glucagon analogs studied were synthesized by automated Fmoc/t-Bu solidphase peptide synthesis and are summarized in Table 1. Our initial selection criteria emphasized aqueous solubility at pH 7 and in vitro potency as assessed by a luciferase-based glucagon reporter assay [Table 2 and Figure 2].17 The starting point of our structure-activity study was Gcg[Aib16] (2), an analog previously demonstrated as possessing enhanced biophysical stability relative to the native hormone.17

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Table 1. Glucagon analogs.

Compound

Analogs modifications

Rt [min]

Th. MW

Exp. MW [H+]

LC-MS ions [m/z]

1

native Gcg

11.5

3482.8

3483.2

871.5; 1161.7; 1742.2

2

Aib16

11.8

3480.8

3481.0

870.9; 1161.0; 1741.2

3

3-Pal6, Aib16

11.5

3481.8

3482.2

871.4; 1161.1; 1741.8

4

3-Pal10, Aib16

11.4

3465.8

3465.9

867.0; 1156.0; 1733.8

5

3-Pal13, Aib16

11.5

3465.8

3465.9

867.1; 1155.8; 1733.3

6

4-Pal13, Aib16

11.4

3465.8

3466.2

867.3; 1155.9; 1733.9

7

3-Pal10,13, Aib16

11.0

3450.8

3450.7

863.4; 1151.0; 1725.8

8

4-Pal10,13, Aib16

11.1

3450.8

3451.2

863.6; 1151.0; 1726.1

9

3-Pal6,10,13, Aib16

10.9

3451.7

3452.6

863.7; 1151.5; 1727.2

10

4-Pal6,10,13, Aib16

10.8

3451.7

3452.2

863.7; 1151.3; 1726.9

11

3-Pal6,10,13, Aib16, 3-Pal22

8.9

3452.7

3453.3

864.0; 1151.7; 1727.4

Peptides were analyzed and characterized by LC-MS (1260 Infinity-6120 Quadrupole LC-MS, Agilent) on Kinetex C8 (4.6 x 75 mm, 2.6 µm, Phenomenex) with 0.05 % TFA/H2O and 0.05 % TFA/CH3CN as eluents employing 5 % B to 70 % B in 15 min gradient with 2.5 min delay; UV detection with λ = 214 nm. Purity based on UV absorbance was determined to be ≥ 95%.

Replacement of Phe6, Tyr10 or Tyr13 correlated with increased solubility for both the 3Pal and 4-Pal substituted analogs. While single substitutions increased solubility to more than 1

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mg/mL [Table 2], double and triple substitution increased it to more than 15 mg/mL and the trend was also reflected in reduced HPLC elution times [Table 1]. The biopotency assessment within the family of analogs prepared indicated that mono, di and even tri-substituted analogs of either 3-Pal or 4-Pal were of picomolar potency. Comparatively, the tetra-substituted analog 11 exhibited poor potency [Table 2, and Figure 2].

Figure 2. Illustration of dose-dependent stimulation of cyclic AMP in Glucagon Receptortransfected cells in response to activation with glucagon (1) and analogs 2-11 [Table 2].

Table 2. Solubility and bioactivity of glucagon analogs. % bioactivity of Gcg

Compound

Solubility [mg/mL]

EC [pM] (SDV)

n

1

< 1

21.2 (13.7)

28

2

< 1

39.9 (11.3)

10

53

3

> 1

17.0 (1.9)

5

125

4

> 1

26.8 (3.1)

3

79

5

> 1

37.9 (17.3)

3

56

6

> 1

33.9 (14.3)

3

63

7

> 15

35.9 (19.3)

7

59

8

> 15

30.9 (10.1)

3

69

50

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9

> 15

71.4 (52.8)

24

30

10

> 15

84.3 (33.4)

3

25

11

> 15

2325.0 (1976)

7

1

n = number of bioassay experiments performed per each analog

The Pal analogs were subjected to more rigorous aqueous solubility studies which examined sustained behavior at 4 oC, starting at a concentration of 1, 5 and 10 mg/mL for an extended time interval. Analogs 8-10 and 11 (data not shown) maintained full solubility under all conditions through 7 days of testing, while all other remaining analogs were less robust with varying levels of diminished performance [Figure 3 and S1].

Figure 3. Change in solubility of Pyridyl-alanine-modified glucagon analogs (Table 1) after incubation at 4 oC. Solubility for peptides 1-6 represents maximal concentration. Initial solubility is shown in red, concentration of peptide in solution after 48 hours at 4 oC in green, concentration after 1 week at 4 oC in blue. The black marked line represents pharmaceutically relevant concentration for injectable emergency glucagon formulation.

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We prioritized analog 9 in our subsequent development studies given its physical, chemical and biochemical properties. Analog 9 proved resistant to fibrillation as measured by the Thioflavin-T fluorescence method using native glucagon and insulin as positive controls [Figure 4]. The single 3-Pal substituted analogs 3, 4 and 5 further diminished the propensity of the native hormone 1 to form fibrils in acidic buffer above and beyond the sizable improvement derived through the single Aib16 substitution, analog 2.

Figure 4. Fibrillation of 3-Pal, Glucagon analogs (Table 1) as measured by fluorescence of Thioflavin-T. (* symbolize analogs 1-5 in 0.1N HCl, while analogs 7 and 9 in aqueous sodium phosphate buffer pH 7.4)

The durability of biological potency as assessed by in vitro methods coupled with measurement of chemical stability by LC-MS at pH 5, 6 and 7 over a four-week period is reported in Figures 5, 6 and S2. Analog 9 proved extremely consistent with little apparent

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change in physical, chemical or biological properties, and 82% quantified after 4 weeks incubation at pH 7 and elevated temperature.

Figure 5. Assessment of change in the in vitro activity of analog 9 incubated in aqueous sodium phosphate buffer at pH 5, 6 and 7, after one and four weeks. Results are expressed as percent change in EC50 values as determined from experimental results shown in Figure S2.

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Figure 6. Assessment of chemical stability by analytical HPLC of compound 9 incubated in phosphate buffer at pH 7, after one and four weeks.

The definitive evaluation of the pharmacological performance of analog 9 was obtained through in vivo studies in rats and pigs. The initial study involved lean rats which were administered escalating doses of native glucagon (1) [Figure S3A] or analog 9 [Figure S3B] with a fixed dose of insulin that was capable of inducing hypoglycemia. The same protocol was repeated comparing analog 9 with native glucagon (1) in the same animal cohort [Figure 7A] to more appropriately assess comparative pharmacodynamics. The groups treated with either native hormone 1 or analog 9 responded similarly relative to the vehicle control group. There was a prevention of insulin-induced glucose lowering at all time points for 60 min following administration, with a slightly elevated response to analog 9 at the earliest time points. To further assess the comparative effect, an additional rat study was conducted in the absence of insulin, where only the speed and magnitude of glucose elevation was measured. The results demonstrate that administration of analog 9 induces an equally rapid onset of action and once

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again a slightly greater elevation of plasma glucose relative to native glucagon (1) [Figure 7B]. The glucagon effect in the analog and native hormone rapidly diminishes following the peak at approximately 30 min [Figure 7A]. This rapid onset and decline of the glucagon effect corroborates the sizable difference in glucose observed in the first hour relative to the next two hours when administered simultaneously with insulin [Figure 7A].

Figure 7. Glucagon (1) and analog 9 effect on change in blood glucose level in lean non-diabetic rats. (∆BG = change in body glucose in mg/dL)

A final measure of the pharmacological properties of analog 9 relative to native glucagon was performed in diabetic pigs, where once again the ability to minimize insulininduced hypoglycemia was investigated [Figure 8]. In this experiment diabetic pigs (4 per group) were treated with insulin detemir (Levemir®), a long-acting insulin analog to lower their blood glucose level by more than 100 mg/dl. Four hours following the insulin injection an identical 10

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nmol/kg dose of either analog 9 solubilized in a physiologically-buffered saline or native glucagon dissolved in the conventional dilute hydrochloric acid was administered subcutaneously. The progressive insulin-induced decrease in blood glucose was reversed to reach a new nadir within 60 min that steadily began to diminish as the short-acting glucagons cleared and the longer-acting insulin effect persisted. As previously shown in rats, glucagon analog 9 demonstrated robust pharmacology that was comparable to the native hormone in magnitude, onset and duration of action.

Figure 8.

Change in glucose level after subcutaneous administration of glucagon (1)

(10 nmol/kg) formulated in 0.1N HCl and analog 9 (10 nmol/kg) formulated in 50 mM sodium phosphate with 150 mM sodium chloride pH 7.4 buffer when administered to diabetic pigs (n = 4) 240 minutes following glucose lowering induced by a subcutaneous injection of a long-acting insulin analog (detemir, 0.5 U/kg) at time 0.

DISCUSSION

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This work is an extension and elaboration of what initially seemed a relatively straightforward objective to identify a safer, more convenient delivery of a life-saving drug at the time of severe hypoglycemia. Despite the huge investment in developing safer insulins with a lower incidence of hypoglycemia,25 a counterbalancing approach utilizing glucagon had been largely ignored until our initial reports.15-18 Glucagon analog 9 demonstrates the requisite biophysical, chemical and pharmacological properties that typify a medicinal drug candidate. Since nature optimizes for physiological rather than pharmaceutical properties it often provides a native ligand that is not readily suitable for use as a drug. Glucagon represents an example of a native hormone which is poorly soluble in aqueous formulations and prone to chemical and physical degradation. These characteristics pose a serious problem during a hypoglycemic episode, since the patient is often too compromised to prepare and self-administer a glucagon injection. Analog 9 appears suitable for formulation as a ready-to-use aqueous preparation of sufficient stability and potency to meet the needs of an insulin-dependent diabetic when severe hypoglycemia occurs. The increased solubility of analog 9 and related peptides in Table 1 was achieved through 3- or 4-Pal substitution at positions 6, 10 and 13, a strategy that preserves the aromatic topology of the N-terminal helix, which is required for receptor recognition.17, 31 While these substitutions subtly reduced in vitro potency relative to native glucagon, the in vivo potency of analog 9 was equivalent in rats and pigs. While glucagon pharmacodynamics is a direct function of pharmacokinetics, direct measurement of the latter was not made and remains an objective for continued study. We propose this “hydrophilic aromatic” approach as an alternative method and supplement to the more established strategy of solubility enhancement through

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isoelectric point adjustment. The planar pyridine heterocycle is uniquely suited to mimic the steric features of a benzene ring, yet provides superior hydrogen bonding capability by virtue of the electronegative character of its nitrogen atom and a strong, permanent dipole (2.21D).26 Notably, among planar heterocycles, pyridine possesses the highest hydrogen bond acceptor strength as measured by its pKBHX value of 1.86.27 These characteristics account for the fact that pyridine, in contrast to benzene is freely soluble in water. Pyridine’s excellent solubility characteristics are realized even in its un-protonated form at physiological pH, well above its conjugated acid’s pKa of 5.23. The use of pyridine as a phenyl isostere has extensive precedent in small molecule medicinal chemistry28 where its favorable properties likely account for the fact that among marketed drugs containing at least one heterocycle, pyridine was the most common one at approximately 25 %.29 Surprisingly, the pyridine modification has only been used in rare instances in peptide medicinal chemistry, perhaps due to a reluctance to use nonnative residues or the belief that a small structural change would be unlikely to alter the solubility characteristics of a macromolecule. In addition to glucagon, a number of therapeutically relevant peptides and proteins suffer from low solubility and a tendency to aggregate.30 These adverse characteristics pose a significant technical hurdle and constitute a high inherent challenge for developmental of peptide drugs. The present findings suggest that substitution of native aromatic residues with 3- or 4-pyridylalanine may offer a versatile means of enhancing solubility and reducing aggregation of therapeutic peptides.

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EXPERIMENTAL SECTION Chemistry. Peptides were prepared by automated (CSBio model CS336X) Fmoc/t-Bu solid-phase methodology starting with pre-loaded Fmoc-Thr(t-Bu)-Wang resin (AAPPtec, Louisville, KY) and HCTU/DIPEA activation. The side chain protecting group scheme consisted of Arg(Pbf); Asp(OtBu); Asn(Trt); Gln(Trt); His(Trt); Lys(Boc); Ser(t-Bu); Thr(t-Bu); Tyr(t-Bu) and Trp(Boc). Fmoc-3-Pal and Fmoc-4-Pal were coupled in the course of automated assembly. All conventional residues were purchased from Midwest Biotech (Fisher, IN), 3- and 4-pyridyl-Lalanines were obtained from AAPPtec (Louisville, KY), HCTU from Peptide International (Louisville, KY), DIPEA from Sigma-Aldrich (St. Louis, MO). Peptides were cleaved from the resin and deprotected by treatment with TFA containing 2.5 % TIS, 2.5 % H2O, 1.5 % methanol, 2.5 % phenol, 0.5 % DODT and 0.5 % of dimethyl sulfide. Peptides were purified by preparative RPHPLC on an Amberchrom-XT20 (21.2 x 250 mm, DOW) and/or Kinetex C8 (AXIA packed, 21.2 x 250 mm, 5 µm, Phenomenex) column with 0.05 % TFA/H2O and 0.05 % TFA/CH3CN as elution buffers. Native glucagon (Eli Lilly and Co., Indianapolis, IN) was re-purified under the above conditions to ensure identical counter-ion content. Purified peptides were analyzed and characterized by LC-MS (1260 Infinity-6120 Quadrupole LC-MS, Agilent) on Kinetex C8 (4.6 x 75 mm, 2.6 µm, Phenomenex) with 0.05 % TFA/H2O and 0.05 % TFA/CH3CN as eluents employing 5 % B to 70 % B in 15 min gradient with 2.5 min delay, HPLC detection – UV absorbance at λ = 214 nm. All peptides were ≥ 95% pure as determined by UV absorbance of the HPLC trace. Peptide’s concentration in solutions was assessed based on UV absorption at λ = 280 nm measured on a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE). Extinction coefficients

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at λ = 280 nm were calculated using on-line Peptide Property Calculator (Innovagen, PepCalc.com). Glucagon Receptor-mediated cAMP Accumulation Assay. The glucagon-induced cAMP production was measured in HEK293 cells over-expressing the glucagon receptor and a luciferase reporter gene linked to cAMP responsive element. The cells were serum deprived for 16 h and then incubated with serial dilutions of glucagon analogs for 5 hours at 37 oC, 5 % CO2 in 96 well poly-D-Lysine-coated “Biocoat” plates (BD Biosciences, San Jose, CA). At the end of the incubation period 50 µl LucLite luminescence substrate reagent (Perkin-Elmer, Waltham, MA) was added to each well. The plate was shaken briefly, incubated for 10 min in the dark and light output was measured on MicroBeta-1450 liquid scintillation counter. Each peptide was run in duplicate per plate. Origin software was used to calculate effective 50 % concentrations (EC50) and standard deviation for each individual experiment using sigmoidal fit with logistic function, Levenberg-Marguardt integration algorithm and statistical weight assigned to each data point. Listed EC50 and SDV values represent average and standard deviation from a minimum of 3 independent assays. Total number of 28 assay experiment was performed with analog 9 being tested 24 times. Solubility. 1 mg or 5 mg (for poly-Pal-substituted analogs) of lyophilized peptide was treated with 200-400 µL of PBS (pH 7.4). Samples were vortexed and sonicated for 10 min then equilibrated at room temperature for 1 hour, following centrifugation at 10,000 rpm for 10 min. Concentration of the peptides in obtained supernatant was determined by measuring UV absorbance at λ = 280 nm. From a highly concentrated stock solution of analogs 7 to 10

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dilution to 10, 5 and 1 mg/mL aliquots were made while evaluating any appearance of precipitate. Concentration in aliquots was re-assessed as previously with UV absorbance. Change in peptide concentration was evaluated after 48 hours and 7 days of storage at 4 oC. Aggregation. Lyophilized peptides were dissolved in appropriate buffer at concentration ~8–9 mg/mL (1-5 in 0.1N HCl; 7 and 9 in 50 mM sodium phosphate, 150 mM of sodium chloride, pH 7.4). Exact concentration was calculated based on UV absorbance at λ = 280 nm and all samples were adjusted to equal concentration of 5 mg/mL. Then all were incubated at 37 °C for 48 hours without agitation, followed by 48 hours at 40 °C with agitation by magnetic stir bar at 300 rmp. Fibrillation was measured according to modified Thioflavin-T fluorescence assay protocol

(http://www.assay-protocol.com/biochemistry/protein-fibrils/thioflavin-t-

spectroscopic-assay). 8 mg of Thioflavin-T (ThT) was dissolved in 10 mL of phosphate buffer (50 mM sodium phosphate, 150 mM of sodium chloride, pH 7.4). Solution was filtered through 0.22 µm syringe filter and stored at 4 oC in dark. Prior to experiment the 0.3 mL of ThT stock solution was further diluted in 15 mL of the phosphate buffer. 5 µL of investigated peptide solution at 5 mg/mL was added to 400 µL of working solution of ThT in phosphate buffer. Solution was incubated for 20 to 30 min. Fluorescence intensity was measured on a Perkin-Elmer LS50B Luminescence Spectrometer (Perkin-Elmer, Waltham, MA) with the following experimental parameters: 350 µL of peptide/ThT solution in sub-micro quartz cuvette [3 x 3 x 3 mm / Z = 9.85 (Hellna GmnG & Co. KG, Mullheim, DE)] excitation λ = 450 nm (slit width 5 nm); emission λ = 482 nm (slit width 10 nm) integration 10 second. Signal was averaged from 4 consecutive points. The fresh solution of insulin in 0.1 N HCl (1 mg/mL; 25 µL in 400 µL ThT working

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solution) and previously aggregated insulin in phosphate buffer (~1 mg/mL; 25 µL in 400 µL ThT working solution) was examined alongside the investigated glucagon analogs. Stability Assay. 2.6 mg of the analog 9 was dissolved in 0.5 mL of phosphate buffer (50 mM sodium phosphate, 150 mM of sodium chloride), and the pH was adjusted to 5, 6 and 7. Samples were vortexed and the fully dissolved peptide solution was filtered over 0.22 µm syringe filter into the sterilized Eppendorf tubes. Concentration was assessed as previously described. Final ~800 µL of 2.0 mg/mL solution was made in sterilized 2 mL glass vial equipped with a sterilized magnetic stir bar. Reference sample at T = 0 was withdrawn and stored at -45 °C, rest of the solutions were capped, sealed with paraffin film and incubated at 37 °C; agitated at 200 rpm. Test samples were withdrawn at 1 and 4 week time point. From withdrawn aliquots LC-MS analyze was run as described previously and samples in DMEM buffer for the bioassay were prepared. Glucagon receptor-mediated cAMP accumulation assay was performed as previously described. Rat Studies. All studies were approved and performed according to the guidelines of the Institutional Animal Care and Use Committee of the University of Cincinnati. Male Wistar rats (Harlan, IN) were housed on a 12 : 12 hours light-dark cycle (8 am - 8 pm lights on) at 22 oC with constant humidity with free access to standard chow (Teklad LM-485) and water, except as noted. Animal’s age and BW: Figure 7A – 15 week-old and 456.1 ± 27 g; Figure 7B – 17 week-old and 492.7 ± 37.5 g; Figure S3A – 17 week-old and 511.8 ± 45 g; Figure S3B – 25 week-old and 552.9 ± 50.7 g. The food was removed at the onset of the light phase, 3 hours prior the intraperitoneal administration of the compounds. The blood glucose level was determined at

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Journal of Medicinal Chemistry

the intervals indicated using a handheld glucometer (Freestyle, Abbot). The statistical analysis of the results obtained in the in vivo experiments was performed using Prism 6.0 h (GraphPad Software, CA) applying One-way ANOVA followed by Dunnett’s tests, using the insulin group as control. P values lower than 0.05 were considered significant (** P