Synergistic metabolic benefits of an exenatide analogue and cholecystokinin in diet-induced obese and leptin-deficient rodents.

AIM: To test the impact of cholecystokinin (CCK) plus either amylin or a glucagon-like peptide-1 receptor (GLP-1R) agonist on metabolic variables in diet-induced obese (DIO) rodents. METHODS: A stabilized acetylated version of CCK-8 (Ac-Y*-CCK-8), selective CCK1 receptor (CCK1R) or CCK2 receptor (CCK2R) agonists, amylin or the GLP-1R agonist and exenatide analogue AC3174 were administered in select combinations via continuous subcutaneous infusion to DIO rats for 14 days, or Lep(ob) /Lep(ob) mice for 28 days, and metabolic variables were assessed. RESULTS: Combined administration of Ac-Y*-CCK-8 with either amylin or AC3174 induced greater than additive weight loss in DIO rats, with the overall magnitude of effect being greater with AC3174 + Ac-Y*-CCK-8 treatment. Co-infusion of AC3174 with a specific CCK1R agonist, but not a CCK2R agonist, recapitulated the weight loss mediated by AC3174 + Ac-Y*-CCK-8 in DIO rats, suggesting that synergy is mediated by CCK1R activation. In a 4 × 4 full-factorial response surface methodology study in DIO rats, a synergistic interaction between AC3174 and the CCK1R-selective agonist on body weight and food intake was noted. Co-administration of AC3174 and the CCK1R-selective agonist to obese diabetic Lep(ob) /Lep(ob) mice elicited a significantly greater reduction in percentage of glycated haemoglobin and food intake relative to the sum effects of monotherapy groups. CONCLUSIONS: The anti-obesity and antidiabetic potential of combined GLP-1R and CCK1R agonism is an approach that warrants further investigation.

The glucagon-like peptide-1-based therapeutics exenatide and saxagliptin did not cause detrimental effects on the pancreas in mice, rats, dogs and monkeys.

AIMS: Recent reports in the literature have suggested that glucagon-like peptide-1 (GLP-1)-based therapies may lead to increased risk of pancreatic pathology leading to chronic pancreatic injury and pancreatic neoplasia. Extensive non-clinical and clinical safety testing was conducted to support the global development of exenatide twice daily, exenatide once weekly and saxagliptin. Our aim was to integrate these non-clinical data obtained with both mechanisms of GLP-1-based drugs to provide complementary data regarding the potential for drug-induced pancreatic safety signals. METHODS: More than 70 regulated non-clinical toxicology studies in rodents and non-rodents were conducted in accordance with International Conference on Harmonisation and US Food and Drug Administration guidance documents, current industry standards, animal welfare regulations and in compliance with Good Laboratory Practice regulations. Treatment duration was up to 2 years in rodents and up to 12 months in non-rodents using high doses representing large multiples of human exposures (up to 130× for exenatide and 2200× for saxagliptin). Comprehensive pancreas assessments involved more than 2400 pancreata from animals exposed to exenatide and over 1700 pancreata from animals exposed to saxagliptin. RESULTS: Neither exenatide nor saxagliptin treatment resulted in drug-related microscopic changes indicative of acute or chronic adverse effects (including neoplasia) in the endocrine or exocrine pancreas, at doses far exceeding the maximum human systemic exposures. CONCLUSIONS: These data substantially add to the weight of evidence supporting the lack of non-clinical drug-induced pancreatic safety signals in animals exposed to GLP-1-based therapies.

Combined antidiabetic benefits of exenatide and dapagliflozin in diabetic mice.

The combined glucose-lowering effect of exenatide and dapagliflozin has not yet been studied. We investigated this combination (single-dose or 4-week dosing) in diabetic ob/ob mice. Vehicle-corrected basal glucose showed greater reduction 1 h following exenatide + dapagliflozin than with exenatide or dapagliflozin alone, and stayed significantly lower for all groups versus vehicle over 3 h. During an oral glucose tolerance test, glucose excursion (30 min post-dose) was significantly lower for exenatide + dapagliflozin versus exenatide or dapagliflozin, or vehicle. Exenatide + dapagliflozin and exenatide, but not dapagliflozin alone, reduced glucose excretion over 24 h versus vehicle. After dosing for 4 weeks, exenatide, dapagliflozin and exenatide + dapagliflozin similarly decreased haemoglobin A1c (HbA1c). Body weight was reduced only with exenatide or exenatide + dapagliflozin. The glomerular filtration rate was similar with exenatide, dapagliflozin and vehicle, and increased with exenatide + dapagliflozin. Optimized combinatorial dosing of these antidiabetic agents may provide additive glucose lowering in type 2 diabetes mellitus.

A novel long-acting glucose-dependent insulinotropic peptide analogue: enhanced efficacy in normal and diabetic rodents.

AIM: Glucose-dependent insulinotropic peptide (GIP) is an incretin hormone that is released from intestinal K cells in response to nutrient ingestion. We aimed to investigate the therapeutic potential of the novel N- and C-terminally modified GIP analogue AC163794. METHODS: AC163794 was synthesized by solid-phase peptide synthesis. Design involved the substitution of the C-terminus tail region of the dipeptidyl peptidase IV (DPP-IV)-resistant GIP analogue [d-Ala(2) ]GIP(1-42) with the unique nine amino acid tail region of exenatide. The functional activity and binding of AC163794 to the GIP receptor were evaluated in RIN-m5F ß-cells. In vitro metabolic stability was tested in human plasma and kidney membrane preparations. Acute insulinotropic effects were investigated in isolated mouse islets and during an intravenous glucose tolerance test in normal and diabetic Zucker fatty diabetic (ZDF) rats. The biological actions of AC163794 were comprehensively assessed in normal, ob/ob and high-fat-fed streptozotocin (STZ)-induced diabetic mice. Acute glucoregulatory effects of AC163794 were tested in diet-induced obese mice treated subchronically with AC3174, the exendatide analogue [Leu(14) ] exenatide. Human GIP or [d-Ala(2) ]GIP(1-42) were used for comparison. RESULTS: AC163794 exhibited nanomolar functional GIP receptor potency in vitro similar to GIP and [d-Ala(2) ]GIP(1-42). AC163794 was metabolically more stable in vitro and displayed longer duration of insulinotropic action in vivo versus GIP and [d-Ala(2) ]GIP(1-42). In diabetic mice, AC163794 improved HbA1c through enhanced insulinotropic action, partial restoration of pancreatic insulin content and improved insulin sensitivity with no adverse effects on fat storage and metabolism. AC163794 provided additional baseline glucose-lowering when injected to mice treated with AC3174. CONCLUSIONS: These studies support the potential use of a novel GIP analogue AC163794 for the treatment of type 2 diabetes.

No evidence of drug-induced pancreatitis in rats treated with exenatide for 13 weeks.

AIMS: The potential association of glucagon-like peptide receptor agonists (GLP-1RAs) with the development of pancreatitis or pancreatic malignancies in patients with diabetes has been suggested. This study evaluated the long-term effects of the GLP-1RA exenatide on pancreatic exocrine structure and function in the Zucker diabetic fatty (ZDF) rat model of type 2 diabetes. METHODS: Rats received subcutaneous twice-daily injections of 0 (control), 6, 40 and 250 µg/kg/day exenatide for 3 months. Clinical signs, body and pancreas weight, food consumption, HbA1c, fasting serum amylase, lipase, glucose and insulin concentrations were evaluated during treatment and after a 28-day off-drug period to assess the reversibility of any observed effects. Morphometric analysis of pancreatic ductal cell proliferation and apoptosis were performed. RESULTS: Plasma exenatide concentrations were several-fold higher than therapeutic levels observed in humans. No exenatide-related effects were observed on clinical signs, lipase concentration, pancreatic weight, pancreatic histology, ductal cell proliferation or apoptosis. Exenatide improved animal survival, physical condition, glucose concentrations and HbA1c, decreased food intake, and increased serum insulin concentration. Total amylase concentrations, although within normal ranges, were slightly higher in exenatide-treated rats; following the off-drug period, total amylase concentrations were comparable in treated and untreated rats. Exenatide-related minimal-to-moderate islet hypertrophy was observed at doses ≥6 µg/kg/day, with dose-related increases in incidence and degree. These changes were still present after the off-drug period. CONCLUSIONS: Chronic administration of exenatide in ZDF rats resulted in the expected metabolic benefits and improved animal survival, with no adverse effects noted on pancreatic exocrine structure and function.

Considerations for successful transplantation of encapsulated pancreatic islets.

Encapsulation of pancreatic islets allows for transplantion in the absence of immunosuppression. The technology is based on the principle that transplanted tissue is protected for the host immune system by an artificial membrane. Encapsulation offers a solution to the shortage of donors in clinical islet transplantation because it allows animal islets or insulin-producing cells engineered from stem cells to be used. During the past two decades three major approaches to encapsulation have been studied. These include intravascular macrocapsules, which are anastomosed to the vascular system as AV shunt; extravascular macrocapsules, which are mostly diffusion chambers transplanted at different sites; and extravascular microcapsules transplanted in the peritoneal cavity. The advantages and pitfalls of these three approaches are discussed and compared in the light of their applicability to clinical islet transplantation. All systems have been shown to be successful in preclinical studies but not all approaches meet the technical or physiological requirements for application in human beings. The extravascular approach has advantages over the intravascular because since it is associated with less complications such as thrombosis and infection. Microcapsules, due to their spatial characteristics, have a better diffusion capacity than macrocapsules. Recent progress in biocompatibility of microcapsules has brought this technology close to clinical application. Critical issues such as limitations in the functional performance and survival are being discussed. The latest results show that both issues can be solved by the transplantation of microencapsulated islets close to blood vessels in prevascularized solid supports.

Porcine neonatal pancreatic cell clusters in tissue culture: benefits of serum and immobilization in alginate hydrogel.

Porcine neonatal pancreatic cell clusters (NPCCs) may be a suitable source of insulin producing tissue for transplantation in diabetic patients. The possible beneficial effect of serum on maturation of NPCCs in vitro is difficult to achieve because of cell clumping, which can be avoided by immobilization in alginate hydrogel matrix. Collagenase treated pancreata, cultured for 4 days, formed NPCCs that were embedded in alginate cross-linked with CaCl2 and cultured in modified Ham's F10 medium with 10% fetal calf serum (FCS) for 10 days. NPCCs cultured as suspension in F10+ with 0.5% bovine serum albumin or with 10% FCS were used as control. To prevent the aggregation when cultured with serum, NPCCs were kept as a very diluted suspension. At the beginning and end of the culture, samples were taken for insulin and DNA content and immunostained for beta and non-beta cells. The culture of NPCCs immobilized in alginate resulted with 3-fold increase in insulin content and 9-fold increase in insulin/DNA ratio. Histology revealed evident increase of number of insulin- and other hormone-positive cells compared with the control. Even though 2 weeks in culture resulted in impaired glucose-induced insulin release, the amount of insulin secreted by clusters cultured in the presence of serum was 4-fold higher than in serum-free conditions. After transplantation, NPCCs retrieved from alginate reversed hyperglycemia similarly to NPCCs cultured in standard conditions. In conclusion, this study shows the feasibility of in vitro immobilization of NPCCs in alginate three-dimensional matrix, allowing cell clusters to be cultured at least two times higher density compared with culture in suspension.

C-peptide responses after meal challenge in mice transplanted with microencapsulated rat islets.

AIMS/HYPOTHESIS: This study aimed to assess a response of microencapsulated rat islets to a meal challenge after being transplanted intraperitoneally into diabetic mice. METHODS: Microencapsulated rat islets or control naked syngeneic mouse islets were transplanted intraperitoneally into mice with streptozotocin-induced diabetes. Meal challenges were done 3, 6 and 9 weeks after transplantation. Glucose-induced insulin secretion from microencapsulated islets before and after transplantation was assessed in vitro. RESULTS: Within the first week, all animals transplanted with either microencapsulated rat islets or with syngeneic murine islets became normoglycaemic (< 11 mmol/l). At 4 and 6 weeks, body weight was less than normal in the non-diabetic control mice. Mice with the encapsulated rat islets had lower fasting glucose concentrations and more rapid glucose clearance after a meal challenge than the control mice. The group of mice with transplanted syngeneic islets had similar glucose profiles to control mice, except for slightly accelerated glucose clearance. The C peptide responses of mice with either microencapsulated or naked islets were clearly lower than the controls. An increase of C peptide appeared as early as 20 min in the plasma of the group with encapsulated islets, but this was considerably slower than in the other two groups. Microencapsulated rat islets retrieved 9 weeks after transplantation did not lose their ability to respond to glucose, but their output was less than half of the pretransplant control islets. CONCLUSION/INTERPRETATION: The delivery of C peptide and presumably the accompanying insulin are delayed by restrictions of the capsules and the peritoneal location. However, this delay in reaching peripheral target organs does not prevent microencapsulated grafts from efficiently clearing glucose after a meal.

Enhanced maturation of porcine neonatal pancreatic cell clusters with growth factors fails to improve transplantation outcome.

BACKGROUND: Porcine neonatal pancreatic cell clusters (NPCC) are a potential source of islet tissue for clinical transplantation. They can normalize glycemia after transplantation, although after a relatively long (several weeks) period of time, possibly due to the immaturity of the tissue. METHODS: One week after isolation NPCCs were immobilized in alginate hydrogel to be cultured for 2 more weeks in the presence of different growth factors, which were applied individually or in various combinations. Their effect was assessed by measuring DNA and insulin content, and expression of islet genes by reverse transcriptase-polymerase chain reaction. Enhanced maturation of NPCCs was also evaluated after transplantation in streptozotocin-diabetic mice. RESULTS: A combination of fetal calf serum, insulin-like growth factor-I, nicotinamide and sodium butyrate in NPCCs media from day 7 to day 21 resulted in increased insulin/DNA content and higher expression of insulin, somatostatin, GLUT2 and Nkx6.1 genes. NPCCs cultured under the same conditions from day 3 to day 12 were transplanted into diabetic mice. Control mice were transplanted with NPCCs cultured in parallel in the presence of nicotinamide, but with no serum, insulin-like growth factor-I or butyrate. Normoglycemia was achieved at the same rate in both groups. Plasma porcine C-peptide (week 6) and graft insulin content (week 20) were also similar in both groups. CONCLUSIONS: Increased insulin content of NPCCs was achieved in vitro by addition of fetal calf serum, insulin-like growth factor-I, nicotinamide, and sodium butyrate, but this increase did not translate into a faster achievement of normoglycemia after transplantation, which suggests that there is a time frame required for complete maturation that is difficult to alter.

In vitro cultivation of human islets from expanded ductal tissue.

A major obstacle to successful islet transplantation for both type 1 and 2 diabetes is an inadequate supply of insulin-producing tissue. This need for transplantable human islets has stimulated efforts to expand existing pancreatic islets and/or grow new ones. To test the hypothesis that human adult duct tissue could be expanded and differentiated in vitro to form islet cells, digested pancreatic tissue that is normally discarded from eight human islet isolations was cultured under conditions that allowed expansion of the ductal cells as a monolayer whereupon the cells were overlaid with a thin layer of Matrigel. With this manipulation, the monolayer of epithelial cells formed three-dimensional structures of ductal cysts from which 50-to 150- micrometer diameter islet-like clusters of pancreatic endocrine cells budded. Over 3-4 weeks culture the insulin content per flask increased 10- to 15-fold as the DNA content increased up to 7-fold. The cultivated human islet buds were shown by immunofluorescence to consist of cytokeratin 19-positive duct cells and hormone-positive islet cells. Double staining of insulin and non-beta cell hormones in occasional cells indicated immature cells still in the process of differentiation. Insulin secretion studies were done over 24 h in culture. Compared with their basal secretion at 5 mM glucose, cysts/cultivated human islet buds exposed to stimulatory 20 mM glucose had a 2.3-fold increase in secreted insulin. Thus, duct tissue from human pancreas can be expanded in culture and then be directed to differentiate into glucose responsive islet tissue in vitro. This approach may provide a potential new source of pancreatic islet cells for transplantation.

Reversal of hyperglycemia in mice after subcutaneous transplantation of macroencapsulated islets.

BACKGROUND: Macroencapsulated islets can reverse hyperglycemia in diabetic animals when transplanted i.p. or into the fat pad. The s.c. space is an attractive site for such transplantation because macrocapsules can be implanted with local anesthesia and be easily removed or reloaded with fresh islets. METHODS: Immunoprotective 20 microl ported devices were transplanted under the skin of Streptozocin-diabetic nude mice. Devices were loaded with 1200 rat islets in culture medium or in alginate. Empty devices were implanted for 2 weeks and then loaded with islets. Normal mice and mice with islets transplanted under the renal capsule or under the skin were used as controls. Seven weeks after transplantation, an intraperitoneal glucose tolerance test (IPGTT) was performed, followed by implant removal. RESULTS: Three weeks after transplantation, normal blood glucose levels were observed in all animals. Compared with those of normal controls, IPGTTs showed accelerated blood glucose clearance in mice transplanted with islets either within devices or beneath the kidney capsule. Fasted transplanted mice were hypoglycemic before glucose injection and 2 hr later. After removal of the implants, all recipient mice returned to hyperglycemia. Histological evaluation revealed viable islet cells and a network of close vascular structures outside the devices. CONCLUSIONS: Macroencapsulated islets transplanted into the s.c. space were able to survive and regulate blood glucose levels in mice. The observed differences in glucose metabolism between normal and transplanted mice may be attributed to the site of transplantation and to the use of rat islets, which have a different set point for glucose induced insulin release.

Differentiation and expansion of beta cell mass in porcine neonatal pancreatic cell clusters transplanted into nude mice.

Neonatal porcine pancreas has considerable capacity for growth and differentiation, making it an attractive potential source of islet tissue for xenotransplantation. Pancreases from 1-3-day-old newborn pigs were digested with collagenase and cultured for 8 days. The resulting cellular aggregates are called porcine neonatal pancreatic cell clusters (NPCCs). The mean yield of NPCCs from a newborn pig was 28,200 +/- 1700 islet equivalents. Cytokeratin 7 (CK7) was used as a marker for the immunostaining of pancreatic duct cells. In neonatal pancreas, 18% of the insulin-positive cells co-stained for CK7, thus being protodifferentiated. NPCCs also contained protodifferentiated cells; insulin/PP and insulin/somatostatin co-stained cells were more common than insulin/glucagon cells. Between 1 and 8 days of culture, the DNA content of the NPCCs fell to 16% and the insulin content to 33% of the starting value, mainly due to the preferential loss of exocrine cells. Transplantation of 2000 or 4000 NPCCs into diabetic nude mice typically normalized glucose values in 10-20 weeks. Mice with successful grafts had lower fasting blood glucose levels than normal mice and accelerated glucose clearance after an i.p. glucose load. The starting NPCCs consisted of 17% insulin-staining cells, but the grafts of mice with reversed diabetes consisted of 94% beta cells, with some co-stained for CK7, indicating that the grafts still contained immature cells. The mass of insulin-producing cells rose from 0.22 +/- 0.08 mg 1 week after transplantation to 4.34 +/- 0.27 mg in mice sacrificed at 27-35 weeks. In summary, NPCCs contain mostly islet precursor cells, which when transplanted into nude mice undergo striking differentiation and beta cell expansion.

[Experiments with pancreas islet xenotransplantation]. / Doswiadczenia w ksenotransplantacji wysp trzustkowych.

Different methods of human, porcine and rat pancreata digestion Langerhans islets purification, immuno-isolation and cryopreservation were compared. The results obtained were assessed in vitro and in vivo. The longest concordial xenograft survival was observed after transplantation of rats islets immunoisolated by Sun's method to streptozotocin diabetic mice. Due to its simplicity and lesser time consumption using Kriomedpol machine was recommended.

Reversal of hyperglycemia in streptozotocin diabetic mice by xenotransplantation of microencapsulated rat islets.

Rat pancreatic islets were immunoisolated within alginate capsules with additional polyethyleneimine-protamine-heparin highly biocompatible membrane. Perifusion study in vitro demonstrated satisfactory similarities between the insulin release profiles of encapsulated and free islets. Concordant xenotransplantation of microencapsulated rat islets significantly prolonged mean time of restored normoglycemia (46 +/- 15 days) in streptozotocin-diabetic BALB/c mice recipients comparing to uncoated grafts (7 +/- 2 days).

Porcine neonatal pancreatic cell clusters (NPCCs): a potential source of tissue for islet transplantation.

This is a short review of porcine neonatal pancreatic cell clusters (NPCCs) which might eventually be useful for beta cell replacement therapy in people with diabetes. The current success with islet allograft transplantation is reviewed and is problematic because only partial success has been obtained and the shortage of human islet tissue means that only a small fraction of people with diabetes would be able to benefit. For these reasons there is considerable interest in xenotransplantation, with pigs being a particularly attractive source. The relative merits of early fetal, late fetal, neonatal and adult porcine tissue are discussed. Neonatal tissue has several attractive features, with their hardiness and potential for growth being especially noteworthy. NPCCs are harvested after digested and dispersed clumps of cells are kept in culture for 7 days. The NPCCs consist mainly of duct cells, protodifferentiated cells and mature endocrine cells. The protodifferentiated cells are either double or triple stained for insulin, cytokeratin 7, glucagon, pancreatic polypeptide, or somatostatin. When transplanted into diabetic nude mice it usually takes weeks before glucose levels are normalized, and during that time differentiation and growth of the graft can be observed. Potential strategies for controlling xenograft rejection are mentioned, with these being immunosuppression, induction of tolerance, immunobarrier devices, and gene transfer approaches.

Comparison of two methods of pancreas islets immunoisolation.

The efficacy of two methods of Langerhans islets immunoisolation was compared. For this purpose the function of islets encapsulated with alginate/polyethylenimine/protamine/heparin (APPH) or with alginate/poly-L-lisine/alginate (APA) membranes was assessed: in vitro according to their survival and response to glucose challenges, and in vivo according to their capability to provide sufficient insulin delivery to maintain normal fasting blood glucose following xenotransplantation to streptozotocin diabetic mice. In vitro insulin secretion and the response to glucose challenge of APPH and APA encapsulated islets were comparable to free islets. In vivo intraperitoneal concordant xenotransplantation of APA encapsulated rat islets reversed the diabetic state of streptozotocin diabetic mice for a longer period, than APPH islet grafts. This study clearly demonstrated the inadequacy of in vitro methods in the prediction of in vivo results of islets transplantation.