4.6 In vivo studies in mice

All experimental procedures were conducted under protocols approved by the Duke Institutional Animal Care and Use Committee (IACUC). Constructs were endotoxin purified prior to injection by passing the solution through a sterile 0.22 µm Acrodisc filter comprised of a positively charged and hydrophilic Mustang® E membrane (Pall Corporation). Mice were group housed in a room with a controlled photoperiod (12 h light/12 h dark cycle) and allowed at least 1 week to acclimate to the facilities prior to that start of procedures. Animals had ad libitum access to water and food. Mice were fed a standard rodent diet (LabDiet 5001) unless otherwise indicated and observed daily.

For evaluating the ELP series with variable T_t or MW, 6-week old, C57Bl/6J male mice (stock number 000664, Jackson Laboratories) were purchased. Mice (n=5 to 6 per treatment group) were immediately placed on a 60 kcal% fat diet (Research Diets D12492) and allowed 1 week to acclimate to facilities. Treatment groups were randomized. On day 0, initial body weight and blood glucose were measured; then mice were injected SC with GLP1-ELP fusions (700 nmol/kg, 200–500 µM) or an equivalent volume of PBS kept on ice. A small nick of the tail vein was made with a lancet. The first drop of blood was wiped clean and the second drop was measured using an AlphaTrak2 handheld glucometer (Abbott), which measures blood glucose using the glucose oxidase method. Glucose measurements were taken periodically throughout day 0 and then every 24 h post-injection.

For efficacy and dose response studies in diet-induced obese (DIO) subjects, 6-week old male C57Bl/6J mice (n=5) were maintained on the high-fat diet for 11 weeks prior to treatment. For the dose response study, GLP1-ELP concentration was kept constant and injection volume was adjusted for the various treatment groups. For efficacy studies in strains with more progressed diabetes, male mice homozygous for the spontaneous Lep^ob mutation (ob/ob, strain 000632) or the Lepr^db mutation (db/db, strain 000697) were used. These mice were treated with the same dose and measured as previously described.

6-week old male C57BL/6J mice (n=5, stock number 000664) placed on a 60 kcal% fat diet were injected with GLP1-ELP every 6 days for 8 weeks (700 nmol/kg, 200 µM). A1C was measured at the end of the study using a handheld A1cNow meter (Bayer), which uses a photometric immunoassay platform. 4-week old ob/ob mice (n=4, stock number 000632) were purchased from Jackson Laboratories and injected with GLP1-ELP every 7 days (700 nmol/kg, 200 µM). Blood glucose and weight were measured periodically and glycosylated hemoglobin (%HbA1c) was measured using a monoclonal antibody agglutination reaction automated by a DCA Vantage Analyzer (Siemens).

During the first week of the long-term study in ob/ob mice (n=4), two IPGTTs were performed. Mice received a single, SC injection of GLP1-ELP_opt or equivalent volume of PBS on day 0. At 66 h post-injection, mice were fasted for 6 h and then challenged with an i.p. injection of 1g/kg, 10 w/v% sterile glucose (Sigma-Aldrich). At 72 h post-injection, blood glucose was measured at 0, 10, 20, 40, 60, 90, 120, and 170 m after glucose administration. This procedure was repeated at 144 h post-injection for a day 6 IPGTT. The glucose meter is unable to read glucose levels below 20 or above 750 mg/dL. For measurements where the meter indicated “HI”, maximal values of 750 mg/dL were substituted to enable statistical analysis.

GLP1-ELP_opt and a soluble control, GLP1-ELP_sol with the same number of VPGXG repeats (310 µM), as well as custom ordered modified GLP1 peptide (75 µM, Anaspec), were radiolabeled with Na¹²⁵I (Perkin Elmer) using the Chizzonite indirect method⁶⁷ for protein iodination to minimize oxidative damage to the peptide⁶⁸. Briefly, in IODOGEN tubes (Thermo Fisher) pre-wetted with 50 µL of PBS, Na¹²⁵I was added to each construct at a molar ratio of 1:250 iodine to GLP1. After 5–10 m on ice, the oxidation reaction was quenched with the addition of 10 µL 0.1% trifluoroacetic acid (TFA). Free Na¹²⁵I was removed using overnight dialysis in 500 mL sterile PBS (Sigma). The final activities were 1.18 µCi/µL free peptide (37.5 µM), 0.49 µCi/µL GLP1-ELP_sol (200 µM), and 0.72 µCi/µL GLP1-ELP_opt (200 µM). An identical procedure was performed using non-radioactive NaI, after which concentrations were determined by measuring 280 nm absorbance on a NanoDrop 1000 and successful iodination was confirmed with matrix-assisted laser desorption and ionization mass spectrometry (MALDI-MS) on trypsin digested samples (SI Fig 1). For the tryptic digest, samples were diluted to 25 µM in 50 mM ammonium bicarbonate and incubated with 0.2 µg MS grade trypsin (ThermoFisher Scientific) for 4 h at 37°C. Samples were diluted 1:10 in 10 mg/mL 4-hydroxycinnamic acid (HCCA) matrix prior to analysis with a DE-Pro MALDI-MS (Applied Biosystems).

6-week old, male ob/ob mice (n=4) were treated with a single, SC injection of radiolabeled GLP1 (30 nmol/kg), GLP1-ELP_sol (700 nmol/kg), or GLP1-ELP_opt (700 nmol/kg) and imaged with a U-SPECT-II/CT imaging system using a 0.350 collimator (MILabs B.V., Utrecht, Netherlands) courtesy of G. Al Johnson at Duke University’s Center for In Vivo Microscopy (CIVM). Anesthesia was maintained with a 1.6% isoflurane feed at an O₂ flow rate of 0.6 L/m. All 20 m SPECT images were reconstructed at 0.2 mm voxel size with MILabs proprietary software without decay correction and centered on the ¹²⁵I photon range (15–45 keV). These reconstructed SPECT images were then registered with their corresponding CT scans (615 µA, 65 kV) to provide spatial alignment for anatomical reference. For each subject, at each time point, the total photon intensity was calculated using ImageJ for 1) the entire image and 2) for an ROI selected to contain the depot and ensure exclusion of the thyroid, bladder, and pancreas. Depot retention was calculated by normalizing the depot ROI intensity to that subject’s 0h full image intensity.

The same mice from the μSPECT-CT study treated with radiolabeled constructs were also used to study depot pharmacokinetics in parallel. Following SC treatment, total body activity was measured using an AtomLab 400 dose calibrator (Biodex) and 10 µL of blood was collected from a tail vein nick into 90 µL of 1000 U/mL heparin. Total body activity measurements and blood draws were repeated at 0.75, 2, 4, and 6 h post-injection and every 24 h thereafter out to 144 h in the GLP1 and GLP1-ELP_sol groups and to 240 h in the GLP1-ELP_dep group. Upon completion of the study, mice in the GLP1-ELP_dep group were euthanized and dissected. Local SC injection site skin and fat were excised in addition to distal skin, distal fat, flank muscle, heart, lungs, thyroid, liver, pancreas, spleen, stomach, and kidneys. A separate cohort of mice was used to measure the pharmacokinetics of the same constructs following a 10 nmol/kg IV bolus injection of radiolabeled drug diluted to 1 µM to prevent phase transition. 10 µL of blood was collected into 1000 U/mL heparin solution at 40 s, 10 m, 45 m, 1.5 h, 3 h, 6 h, 12 h, 18 h, 24 h, 36 h, 48 h, and 54 h post-injection.

Radioactivity of the dissected organs and all blood samples was quantified with a Wallac 1282 Gamma Counter (Perkin Elmer). To calculate the half-life, raw CPM values of blood samples were plotted against time and fit to a one-phase exponential decay function in GraphPad Prism 6 using data points in the elimination phase of the PK curve for the IV study and after t_max for the SC study. These curves were fit for each subject individually because of slightly variable time points. The parameters presented are the average values within each treatment group and the standard error of the mean (SEM).

For quantification of circulating drug concentrations, the gamma count for each sample was converted to nanomolar concentration using a set of standards and then dividing by the blood sample volume (10 µL). For the IV data, t_{1/2, elim} was calculated as ln(2)/k_e where k_e was found by fitting a line of exponential decay to the elimination phase of the PK curve generated from raw CPM values (45 m to 48 h range for the fusions and 2 min to 1.5 h range for the peptide). For the SC PK data, the same fit was used covering time points after C_max had been reached to calculate the biological half-life (t_{1/2, biol}), which accounts for the controlled release and slowed absorption from the SC route of administration. In order to quantify C_max and compare the drug AUC for all three constructs, the detected counts per minute (CPM) were converted to molar concentration using a standard curve built from aliquots of each injected construct, whose activities and concentrations were known. Bioavailability (F) was calculated as the ratio of dose-normalized AUC_SC to AUC_IV, using the equation: F = (AUC_SC×Dose_IV)/ (AUC_IV×Dose_SC). Clearance (CL) was calculated as (Dose×F)/(AUC) where F = 1 for a bolus IV injection. Although there have been few reports of a minimum effective concentration of GLP1 in mice, we approximated a value (33 nM) based on a study of how GLP1’s insulin stimulating effects are dependent on intravenous dose⁶⁹ in conjunction with a study that involved measuring plasma GLP1 levels after intravenous administration⁷⁰. The calculation of this value is discussed in more detail in Supplementary Information. The 40 nM value also correlates with the concentration at which a maximum GLP1 receptor response is observed for fusion treatment in vitro.

5-week old, male ob/ob mice were allowed one week to acclimate to the facilities. On the day of injection, mice were anesthetized with isoflurane and shaved across their backs. Two circles, approximately 12 mm in diameter, were marked bilaterally with permanent marker and injected in the center of the circle at 50 mg/kg or an equal volume of saline. Injections of PBS, GLP1-ELP_opt, or PLGA microspheres 50 µm in diameter (Sigma) were randomized, but stratified to ensure that no mouse received the same injection type bilaterally. After 5 days, the mice were euthanized and the skin was dissected. A 12 mm biopsy punch (Electron Microscopy Sciences) was used to remove the area indicated by the permanent marker. The skin was cut in half and placed in 10% neutral buffered formalin. After paraffin embedding, three 5 µm sections were taken from the midline cut, spaced apart by 50 µm. These sections were stained with hematoxylin and eosin and imaged using a Zeiss laser microdissection and capture microscope equipped with an AxioCam ICc3 color camera. The slides were inspected and analyzed by a certified pathologist who was blinded to the groups.