Dulaglutide Case Study Essay

Dulaglutide is a Glucagon-like peptide-1 (GLP-1) receptor agonist (RA) indicated for the treatment of type 2 diabetes mellitus (T2DM). Dulaglutide was invented by Wolfgang Glaesner, Rohn Lee Millican Jr, and Andrew Mark Vick, employees of Eli Lilly and Company. In the United States dulaglutide is manufactured by Eli Lilly and Company in Indianapolis, Indiana under the trade name Trulicity.

T2DM is a progressive disease that is characterized by the dysfunction of ? pancreatic islet cells that produce insulin, insulin resistance and hyperglucogonaemia, all which contribute to chronic hyperglycemia (Glaesner, et al. 2010). GLP-1 (or incretin) is a hormone secreted by intestinal endocrine cells following a meal. GLP-1 supports glucose homeostasis by stimulating the secretion of insulin from B-islet pancreatic cells. GLP-1 also has been demonstrated to reduce food intake & appetite and inhibit gastric emptying. These effects may be beneficial in weight management and thus the management of T2DM. In patients with type 2 diabetes the effect of GLP-1 and be markedly reduced or absent (Nauck, et. al, 1986).

GLP-1 has limited therapeutic potential due to its short half-life and rapid degradation by the body’s intrinsic dipeptidyl peptidase IV (DPPIV) activity (Drucker & Nauck, 2006). GLP-1 receptor agonists with extended half-lives such as dulaglutide have been shown to be effective in restoring insulin secretion in patients with T2DM. In order to increase the half life of GLP-1 and lower its clearance, GLP-1 was fused to a large carrier moiety. GLP-1 fused to a plasma carrier proteins albumin and the Fc domain of immunoglobulin.

When GLP-1 was fused to albumin in preclinical studies the half life of the GLP-1-albumin complex was ncreased to 10-12 hours. When GLP-1 was fused to the Fc domain of immunoglobulin G the half life of the GLP-1-FC immunoglobulin G complex was increased significantly to 30 hours (Picha, et al. , 2008). The insulinotropic effects of the the endogenous GLP-1 was maintained when it was attached to the Fc fragment of modified immunoglobulin G. In addition, The plasma half-life was improved, clearance was decreased, and a flat dose/response profile was observed with no burst effect (Glaesner, et al. , 2010). This pharmacokinetic profile allows many GLP-1 RAs to be dosed on a weekly regimen. Discovery

In vitro studies Human embryonic kidney 293-EBNA cells were transfected with DNA in a reduced serum medium and incubated. The expression media was loaded and washed using a Protein A Sepharose High Performance column equilibrated in PBS for purification. Pooled fractions of bound GLP-1-Fc were concentrated and then equilibrated in a high performance gel chromatography column. GLP-1-Fc fractions were characterized using SDS-PAGE and mass spectrometry and assessed for concentration (Glaesner, et al. , 2010). Potential T-cell epitopes were identified in silico using an algorithm for T-cell epitope mapping.

The N-terminal 64 amino acids of GLP-1-Fc were identified and binding was predicted to eight MHC-Complex class II alleles that are representative of human populations. Binding was scored and ranked. Antibody dependent, cellmediated cytotoxicity assays were performed with Jurkat Fcylll cells. Chinese hamster ovary-K1_GLPR1 cells were plated on luminescent plates and incubated with various concentrations of GLP-1Fc. Luciferase activity was assayed by incubation with a luminescence reagent and analyzed using a luminometer.

GLP-1 expressing and CAMP responsive HEK 293 cells were analyzed y measuring the activity of reporter genes by incubating with gentamycin and L-glutamine and measuring bioluminescence after the addition of B-luciferase (Glaesner, et al. , 2010). HEK 293 cells that expressed human GLP-1 receptor were incubated with a CAMP responsive Cre/BLAM reporter system that uses Cre recombinase as a reporter of signal transduction (Mattheakis, et al. , 1999). Following incubation the maximal effective concentration values were determined from a dose response curve by adding 0. 00003-3nM dilutions of GLP-1 agonist. Lactamase substrate was then added and florescence was measured (Glaesner, et al. , 2010).

The directly fused DPPIV-protected GLP-1 (V8-GLP-1) analog to human immunoglobulin G hinge reduced in vitro activity by about 95% compared to free V8-GLP-1. This problem was solved by adding linker sequences between the C-terminus of modified GLP-1 analogs and the N-terminus of the IgG hinge. An optimal linker was identified and this linker demonstrated 4-fold greater in vitro potency as compared to V8-GLP-1. This compound was further optimized to reduced potential complement-dependent and antibody-dependent cell-mediated cytotoxicity. IgG1 was replaced with a modified Ig4 to reduce interaction with highaffinity Fc receptors.

This resulted in significant reductions of dose-dependent cytotoxicity in favor of the IgG4 version over the IgG1 version. Further optimization was performed to eliminate half-antibody formation, S228 was mutated to P228. In order to de-immunize the fusion protein the GLP-1 R36G mutation was introduced and the C-terminal K of IgG-Fc was removed. Following optimization dulaglutide had 4-fold greater GLP-1-RA activity comparted to V8-GLP-1 peptide (Glaesner, et al. , 2010). Purified rat pancreatic islets were cultured.

These purified islets were incubated in a salt solution with 2. 8mM to 16. 8 mM. lucose and increasing levels of dulaglutide with or without exendin, a well characterized GLP-1 receptor agonist (Ding, et al. , 2006). Insulin was then measured over a 90-minute period. Insulin secretion was significantly increased with the inclusion of 20 nM dulaglutide at the high dose of glucose (16. 8 mM), and no significant increase in insulin secretion was observed in the low dose of glucose (2. 8 mM). Unmodified GLP-1 caused a 4-fold increase in insulin secretion, whereas dulaglutide caused a 3fold increase in insulin secretion, demonstrating that dulaglutide is nearly as efficacious as GLP-1 in stimulating insulin secretion.

The EC50 of insulin secretion by dulaglutide was observed at 2. 7 NM, while the Emax was observed at 300 nM. The inclusion of exendin reversed the glucose dependent stimulation of insulin secretion that was observed with dulaglutide suggesting that dulaglutide acts on the islet GLP-1 receptor (Glaesner, et al. , 2010). Following results demonstrating increased insulin secretion in the rat islet cells, the test was repeated using pancreatic cells from cynomolgus monkeys. The monkey cells were cultured in RPMI-1640 medium and were starved in EBSS containing 2. mM glucose.

Batches of three islets were incubated in EBSS and 16. 7 mM glucose and increasing levels of dulaglutide with or without exendin. The results from the rat studies corroborated with the results shown in this study (Glaesner, et al. , 2010). A concentration dependent glucose secretion effect of also observed in the monkey islet cells. In vivo studies Sprague-Dawley rats and cynomolgus monkeys were used in order to determine the pharmacokinetics of dulaglutide. Adult male rats received a single subcutaneous dose of 0. mg per kg dulaglutide, and blood samples were collected 1,2,4 and 6 days following administration. A group of 3 monkeys received a subcutaneous dose of 0. 1 mg per kg dulaglutide and blood samples were collected at 0, 2, 4, 12, 48, 72 96, 192, 240, 288 and 336 hours following administration.

Samples from both animals were studies for GLP-1-Fc concentration using ELISA, utilizing antibodies that recognize the N-terminus of GLP-1-Fc and the Fc domain. Optical density of tetramethylbenzidine development was measured and concentrations of GLP-1-Fc were calculated using the four parameter algorithm (Glaesner, et al. 2010). The pharmacokinetic parameters of dulaglutide for rats and monkeys respectively were: t1/2= 38. 2 hours, and 51. 6 hours; Cmax = 179. 7 ng/mL, and 292. 2 ng/mL; Tmax= 24 hours, and 16. 7 hours; Cl = 9. 6 ml/h/kg, and 7. 3 mL/h/kg; VD = 525. 0 ml/kg, and 557. 5 mL/kg; AUCO-00 = 10,537 and 15,207 (Glaesner, et al. , 2010; Jimenez-Solem et al. , 2010). Graded glucose infusion studies were performed in both rats and monkeys. Adult rats were treated with subcutaneous injections of normal saline of dulaglutide at concentrations 0. 3, 1, 3, or 30 nmol/kg increments and fasted for 16 hours.

Rats were then infused with saline for 20 minutes, followed by a low dose of glucose at 50 mg/kg/min for 30 minutes and a high dose of glucose at 150 mg/kg/min for 30 minutes. Blood samples were collected at 20 and 10 and 0 minutes prior to infusion and every 10 minutes following administration for 1 hour. Insulin secretion was significantly increased in rats that received 3 nM and 30 nM concentrations of dulaglutide in the presence of the high dose of glucose, but did not increase insulin secretion at low glucose doses (Glaesner, et al. , 2010).

Fasted monkeys received infusions of glucose immediately following subcutaneous administration of vehicle (PBS) or dulaglutide at 1. 7 nmol/kg and at days 1,5 and 7 following administration. 20% glucose solution (D20W) was infused at 10 mg/kg/min for the first 20 minutes, and then at 25 mg/kg/min for the following 20 minutes. Blood samples were collected at 10 and 0 minutes prior to administration and at 10, 20 30 and 40 minutes following administration. Monkeys administered dulaglutide showed significantly enhanced insulin responses to glucose for at least 7 days (Glaesner, et al. , 2010).

A separate experiment was also performed where monkeys received vehicle or dulaglutide (1. 7 nmol/kg) once per week for 4 weeks and evaluated using the graded glucose infusion method described above four days following the final dulaglutide dose. This experiment also demonstrated increased glucose stimulated insulin secretion 4 days following the final dose (Glaesner, et al. , 2010). PND35 female diabetic mice were administered 10 nm/kg does of dulaglutide once per week for 4 weeks. Blood glucose was measured before each injection at weeks 2, 3, and 4, and at 1 hour after administration at week 1.

Fasting insulin levels were measured on day 0 and day 26 after fasting overnight. Dulaglutide dosed mice showed consistently lower plasma glucose during the period of administration as compared to controls (Glaesner, et al. , 2010). These mice also demonstrated a significant weight loss as compared to controls (Glaesner, et al. , 2010). Favorable pharmacokinetic parameters obtained in pre-clinical development led to the approval of Eli Lilly’s dulaglutide IND (U. S. Food and Drug Administration, 2014). Pharmacokinetic parameters were studied in Phase I healthy volunteers who ere given 0. 1 to 12 mg subcutaneous doses administered 3 weeks apart. The t1/2= 90 hours and the Tmax= 24-48 hours, F= 47-65%, VD = 19. 2 L for a 0. 75 mg dose and 17. 4 L for a 1. 5 mg dose; Cl = 0. 111 L/h for a 0. 75 mg dose and 0. 107 L/h for a 1. 5 mg dose (Jimenez-Solem et al. , 2010). The long half life of dulaglutide gives it a significant advantage. Data obtained in clinical trials suggest that greater stability leads to higher efficacy and a lower frequency the common side effects of nausea and vomiting observed with drugs of this class (Jimenez- Solem et al. , 2010).