EXENDIN-4 IS A 39-amino acid reptilian peptide that

Transcription

1 X/04/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 89(7): Printed in U.S.A. Copyright 2004 by The Endocrine Society doi: /jc Exendin-4 Normalized Postcibal Glycemic Excursions in Type 1 Diabetes JOHN DUPRÉ, MARGARET T. BEHME, AND THOMAS J. MCDONALD Department of Medicine, University of Western Ontario and London Health Sciences, London, Ontario N6A 5A5, Canada Exendin-4 is a reptilian peptide that activates the mammalian receptor for truncated glucagon-like peptide 1 (tglp-1) with relatively prolonged actions. Exendin-4 and tglp-1 can reduce blood glucose levels by stimulating insulin secretion, inhibiting glucagon secretion, and delaying gastric emptying. We tested a range of doses of exendin-4 on postcibal glycemic excursions in nine volunteers with type 1 diabetes, all with negligible endogenous insulin secretion, in paired comparisons with vehicle in at least six volunteers with each of six doses. We established a side effect-free dose and an appropriate antecibal time for sc administration of exendin-4. Subsequently, exendin-4 was administered 15 min before breakfast, EXENDIN-4 IS A 39-amino acid reptilian peptide that activates mammalian receptors for the truncated glucagon-like peptides 1 (7 37) and (7 36 amide) (tglp-1) (1). Both tglp-1 and exendin-4 exert actions to reduce the blood glucose levels in normal subjects and in those with type 2 diabetes by stimulating insulin secretion and inhibiting glucagon secretion in the postabsorptive state and delaying gastric emptying in the postcibal (PC) state (2). The high potency and duration of action of exendin-4 compared with tglp-1 apparently depends on the relatively slow clearance of the reptilian peptide, which is resistant to degradation by dipeptidyl peptidase IV. On account of their known actions and glycemic effects, tglp-1 agonists, including exendin-4, are under evaluation as potential therapeutic agents in the treatment of type 2 diabetes mellitus (2). The PC glycemic effects of the incretins gastric inhibitory polypeptide and tglp-1 in humans have generally been attributed to their insulinotropic actions (3, 4). In type 2 diabetes, the preservation of this action of tglp-1, in contrast to loss of this action of gastric inhibitory polypeptide, leads to interest in the therapeutic potential of tglp-1 as an insulin secretagogue in that condition (5). Our observations in insulin-treated clinical type 1 diabetes (T1D) showed that the PC glycemic action of tglp-1 is also present in subjects with little or no capacity for endogenous secretion of insulin (6 8). We suggested that the major mechanism of this effect of tglp-1 in T1D depends on its action to delay gastric emptying, with consequent pharmacodynamic enhancement of the action of exogenous insulin. Because more prolonged Abbreviations: AUC, Area under the curve; BMI, body mass index; HPP, pancreatic polypeptide; PC, postcibal; T1D, type 1 diabetes; tglp-1, truncated glucagon-like peptide 1 (7 36 amide). JCEM is published monthly by The Endocrine Society ( endo-society.org), the foremost professional society serving the endocrine community. with usual insulin, to eight of the volunteers. Acetaminophen was ingested with the meal as an indicator of gastric emptying. The mean plasma glucose excursion was reduced by 90%, falling into the normal range, after breakfast, whereas plasma pancreatic polypeptide, glucagon, and acetaminophen levels were reduced, and insulin levels were not affected. Thus, normalization of postcibal glycemia was associated with delayed gastric emptying and suppression of glucagon secretion, without increased secretion or blood levels of insulin. We suggest that tglp-1 agonists have therapeutic potential as congeners with insulin in C-peptide-negative type 1 diabetes. (J Clin Endocrinol Metab 89: , 2004) delay of gastric emptying might be advantageous, in the present study, we examined the efficacy of the long-acting tglp-1 agonist exendin-4 in experiments similar to those used earlier with tglp-1. Subjects and Methods Experimental subjects All subjects had clinical T1D, with little or no endogenous insulin response to meals (incremental plasma C-peptide levels 0.10 nmol/ liter). The subjects were considered to be in optimized metabolic control using established programs of intensive insulin therapy (multiple daily sc injections or continuous sc infusion of insulin), which were not modified for the purposes of this study. Nine volunteers (five males and four females) with T1D participated in dose-finding studies. They ranged in age from yr (mean se, yr), in body mass index (BMI) from kg/m 2 ( kg/m 2 ), in duration of diabetes from 4 43 yr (20 4 yr), in insulin dose from U/kg d ( U/kg d), and in hemoglobin A 1C from % ( %). Six or more individuals received each dose of exendin-4. Further paired studies with the selected dose of 0.03 g/kg exendin-4 or vehicle were carried out in eight of the nine volunteers with T1D (five males and three females; the ninth volunteer was not available). These subjects ranged in age from yr (mean se, yr), in BMI from kg/m 2 ( kg/m 2 ), in duration of diabetes from yr (25 4 yr), in insulin dose from U/kg d ( U/kg d), and in hemoglobin A 1C from % ( %). Breakfast tests without exendin-4 were also carried out in six normal volunteers (three males and three females) ranging in age from yr (42 5 yr) and ranging in BMI from kg/m 2 ( kg/m 2 ). Each volunteer gave signed informed consent to protocols approved by the Research Ethics Board for Health Sciences Research Involving Human Subjects of the University of Western Ontario. Procedures In dose-finding studies, abdominal sc injections of vehicle or of 0.01, 0.02, 0.03, 0.04, or 0.06 g/kg exendin-4 were administered with the usual dose of insulin immediately before breakfast, between 0730 and 0830 h after overnight fast. The volunteers were blinded to the nature of the test agent on each occasion, and tests were carried out in varied order at least 7 d apart. For further studies, we selected the dose of

2 3470 J Clin Endocrinol Metab, July 2004, 89(7): Dupré et al. Exendin-4 for Type 1 Diabetes g/kg exendin-4, which was the largest test dose that was free of the subjective effects of mild nausea or abdominal discomfort occasionally reported by the volunteers after higher doses. In the further paired studies, this dose or vehicle was administered 15 min before the meal to allow for the delay of at least 15 min in the onset of effect of exendin-4 on the pancreatic polypeptide response (Fig. 1). The usual dose of insulin was again administered shortly before the meal, according to individual treatment programs. Acetaminophen (1000 mg for body weight 70 kg or 1500 mg for body weight 70 kg) was ingested at beginning the meal, and the blood levels of acetaminophen were determined at intervals for assessment of the rate of gastric emptying. Exendin-4 (Bachem, Torrance, CA) was dissolved in 0.05% human serum albumin in saline, sterile-filtered, tested for sterility and pyrogens, and stored at 20 C. Breakfast meals without test peptide were also taken by the normal volunteers to determine PC glycemic excursions. For subjects with T1D, the components and amounts of food were self-selected according to established diet; each breakfast was identical on all occasions for each subject, and every meal was fully consumed. For the normal volunteers, FIG. 1. Plasma glucose, HPP, and incremental glucagon responses to breakfast with sc injection of varying doses of exendin-4 (E, 0 g/kg; F, 0.01;, 0.02; Œ, 0.03;, 0.04; f, 0.06) with usual insulin in volunteers with T1D with no endogenous insulin secretion. See Table 1 for conversion factors. a standard breakfast containing 75 g carbohydrate was provided. Blood samples were obtained at intervals through 240 min from an iv cannula in a superficial forearm vein, collected in heparinized tubes containing aprotinin, held on ice, processed at 4 C, and stored at 70 C. Blood samples were also collected in nonheparinized tubes, held at room temperature, and processed for serum samples, which were stored at 70 C. Methods Blood glucose levels were monitored with a clinical glucose reflectance meter at all sample times, and plasma glucose concentrations were subsequently determined with a Beckman Glucose Analyzer II (Beckman, Palo Alto, CA). Plasma samples were assayed for pancreatic polypeptide with antiserum 615/1054 B (RE Chance; Eli Lilly and Company, Indianapolis, IN), for glucagon with antibody 04A (RH Unger, Dallas, TX), and for C-peptide with antibody 7309 (Peninsula Laboratories, San Carlos, CA), all with commercial 125 I-peptides. Aliquots of serum stored at 70 C were assayed for free immunoreactive insulin with antibody from P. Wright (Cambridge, UK) after extraction with polyethylene glycol (9). Intraassay variability was less than 8% for all RIAs. Interassay variabilities of 9% for pancreatic polypeptide, 16% for glucagon, 12% for C-peptide, and 24% for free immunoreactive insulin were corrected with standard values for control samples run in each assay. Acetaminophen concentrations were determined in the clinical chemistry laboratory. Data are presented as mean values se. Incremental areas under concentration curves (AUC) were calculated geometrically for stated time periods and time averaged by dividing the total AUC by elapsed times. Statistical significance of differences between mean values was determined by ANOVA, polynomial regression analysis, and two-tailed paired t tests. P 0.05 was considered statistically significant. Results In dose-finding studies, the mean values for time-averaged incremental AUCs of plasma glucose concentrations after breakfast were reduced after injection of exendin-4 at 0 min antecibal breakfast in a dose-dependent manner through 4 h (Fig. 1 and Table 1). Likewise, mean values for AUC of pancreatic polypeptide (HPP) concentrations after breakfast were reduced after the injections in a dose-dependent manner; the mean values for HPP concentration did not differ with dose at 15 min but did vary with dose at 30 min and thereafter to 90 min. Mean values for AUC incremental plasma glucagon concentrations through 240 min and for free immunoreactive insulin concentrations through 240 min were not significantly affected. The only perceived adverse effects were three episodes of symptomatic hypoglycemia after injections of 0.04 or 0.06 g/kg exendin-4 that were perceived by the volunteers and confirmed by reflectance meter determination of the blood glucose level; these episodes were reversed with oral carbohydrate according to clinical practice. Due to this observation and to the delay of at least 15 min in onset of the effect of exendin-4 on the response of HPP, further studies were conducted with 0.03 g/kg exendin-4 administered 15 min before the meal. This was the highest side effect-free dose, and higher doses were not used with this timing of administration of test peptide. After administration of vehicle 15 min antecibal, followed by the usual dose of insulin, the volunteers with T1D had higher mean AUCs for plasma glucose than the normal volunteers given a standard meal (P 0.03; Fig. 2 and Table 2). After administration of exendin-4 according to this protocol, the mean PC excursion of glucose concentration was reduced in T1D patients and was not different from that of the normal

3 Dupré et al. Exendin-4 for Type 1 Diabetes J Clin Endocrinol Metab, July 2004, 89(7): TABLE 1. Time-averaged incremental AUCs for plasma GLU (0 240 min), HPP (0 60 min), GON (0 240 min), and FIRI (0 240 min) Dose ( g/kg) No. of subjects GLU a (mm) HPP a (pm) GON (ng/liter) FIRI (pm) Graded doses of exendin-4 administered sc with usual insulin before breakfast at time 0 min in T1D with no endogenous insulin secretion. GLU, Glucose; GON, glucagon; FIRI, free immunoreactive insulin. Conversion factors: GLU, 1 mm mg/dl; HPP, 1 pm g/ml; GON, 1 ng/liter pg/ml; FIRI, 1 pm U/ml. a Varied with dose of exendin-4 with R subjects. The mean AUC of plasma HPP concentration through 4 h in T1Dpatients given vehicle did not differ from the value for normal volunteers; however, after administration of exendin-4, the corresponding mean excursion was almost completely inhibited through 4 h and thus was smaller than that of the normal volunteers given vehicle (P 0.02). The mean incremental plasma glucagon concentration after breakfast was elevated in T1D subjects compared with normal subjects (P 0.03); this response was reduced after the administration of exendin-4 and then was not significantly different from normal. The mean value of AUC for plasma acetaminophen concentrations through 60 min in T1D subjects given vehicle did not differ from that in normal volunteers, but it was reduced after administration of exendin-4. Again, the mean AUC for free immunoreactive insulin concentration was not significantly affected by exendin-4. Symptomatic hypoglycemia occurred at 120 min in one subject with both exendin-4 and vehicle. It was perceived by the subject and confirmed by the reflectance meter estimates of blood glucose level and was reversed by oral carbohydrate according to the practice of the volunteer on both occasions. None of the blood glucose reflectance meter determinations at any other time in any of the volunteers yielded values of less than 3 mmol. There were no other perceived adverse effects. With respect to the relative efficacy of exendin-4 administered 15 min before the meal, the mean plasma glucose levels through 240 min were lower with 0.03 g/kg exendin-4 given at this time point before breakfast compared with the effect of the same dose given immediately before the meal (Figs. 1 and 2; ANOVA, P 0.001). Likewise, reduction of the mean plasma HPP level was more effective with exendin-4 given 15 min before the meal than when it was given immediately before the meal (ANOVA, P 0.02), and suppression of the mean incremental plasma glucagon level was greater and more prolonged with exendin-4 given 15 min before the meal than with exendin-4 given immediately before the meal (ANOVA, P 0.001). Thus, exendin-4 given 15 min before the meal in volunteers with T1D was much more effective than exendin-4 given immediately before the meal. Discussion Exendin-4 given sc 15 min before breakfast normalized PC glycemic excursions through 4 h after breakfast in volunteers with T1D who had little or no residual insulin secretion and who continued established intensive insulin therapy without modification. This effect was accompanied by suppression of the abnormal increase of mean plasma glucagon concentration that occurred with administration of insulin alone. At the same time, the increase of plasma HPP concentrations, which was normal with administration of insulin alone, was largely suppressed after administration of exendin-4. These effects were associated with delay of gastric emptying manifested in postponement of the increase of the blood levels of acetaminophen by 15 min, with reduction of its increase through more than 1 h. It may be noted that the excursion of acetaminophen levels after the meal was normal in the volunteers with T1D given insulin alone, indicating that they had no apparent abnormality of gastric emptying. The results suggest that the glycemic effect of exendin-4 cannot be attributed to increased exposure of the tissues to insulin, given the lack of endogenous insulin secretion and the absence of evidence of a significant effect of exendin-4 on the clearance of exogenous insulin. This is consistent with the lack of such an effect of tglp-1 given by continuous iv infusion on blood levels of free immunoreactive insulin in earlier studies (6). Thus, among the known actions of exendin-4 that might contribute to its glycemic effect in volunteers with T1D, suppression of endogenous secretion of glucagon and inhibition of gastric emptying were both observed. Studies of the mechanism of meal-induced hyperglycemia in insulin-deficient T1D patients have suggested that a lack of appropriate suppression of endogenous glucose production contributes to excessive delivery of glucose to the systemic circulation (10). It has also been shown that intensive insulin therapy can restore suppression of endogenous glucose production to rates observed in nondiabetic subjects, eliminating this factor in meal-induced hyperglycemia (11). However, in the present study, the use of established intensive insulin treatment programs failed to eliminate the abnormal rise in mean plasma glucagon levels after ingestion of breakfast. Under these conditions, the abnormal rise in the mean incremental plasma glucagon level after the meal was prevented by administration of exendin-4 15 min before the meal; therefore, this effect may have contributed to normalization of the glycemic excursion. Because many subjects with T1D, like those in the present study, exhibit continuing abnormality of the glucagon response to meals under clinical conditions, this effect of exendin-4 has potential therapeutic importance. It should be noted here that, although a hepatic action of HPP supporting inhibition of glucose production by insulin has been proposed (12), glycemic effects of reduction of such

4 3472 J Clin Endocrinol Metab, July 2004, 89(7): Dupré et al. Exendin-4 for Type 1 Diabetes FIG. 2. Glucose, HPP, acetaminophen, and glucagon responses with 0.03 g/kg sc exendin-4 at 15 min (F) or vehicle (E) in eight volunteers with T1D and no -cell function (paired studies) compared with responses in six healthy volunteers (dotted line). Please see Table 1 for conversion factors. action might be obscured by the relative hypoglycemia and/or by the inhibition of glucagon secretion by exendin-4 in the present experiments. In earlier studies with tglp-1, postponement of the response of HPP to meals pointed to delay of gastric emptying as a mechanism of enhanced glycemic control (6 8). We speculated that this allowed establishment of insulin s effects in anticipation of absorption of the meal. The degree of suppression of the initial response of HPP observed in the present dose-finding studies with administration of exendin-4 at 0 min was incomplete compared with that observed with tglp-1 in earlier studies, but it was more prolonged. Under these conditions, reduction of the glycemic excursion was less than the reduction observed with administration of exendin-4 15 min before breakfast, when the HPP response to the meal was effectively prevented. The observations on blood levels of acetaminophen confirm the delayed onset of gastric emptying, which has been inferred in earlier studies from the delay in initiation of the HPP response to the meal, and show continuing inhibition of emptying through an interval of1hormore. Thus, the results of studies using tglp-1 agonists in T1D remain consistent with the suggestion that a major component of their PC glycemic effect is related to delay of gastric emptying. This may be associated with pharmacodynamic enhancement of the efficacy of insulin, as demonstrated when a similar delay in administration of nutrient relative to delivery of insulin enhanced the efficacy of an infusion of insulin, with prolonged reduction of the glycemic excursion (13). Establishment of effective blood levels of exendin-4 through the same interval may also account for the greater efficacy of this peptide in suppression of glucagon responses under these conditions. It has also been suggested that the glucagon-like intestinal peptides can have other metabolic effects tending to reduce blood glucose levels after meals. In studies of PC glycemia, reduction of glycemic excursions during prolonged iv infusion of tglp-1 in volunteers with T1D, with concurrent reduction of iv delivery of insulin by programed infusion, was interpreted as suggesting enhancement of insulin sensitivity (14). However, this conclusion did not take into account the effect of tglp-1 on gastric emptying, which can lead to retention of much of the meal within the stomach through the infusion period (15). Further studies of insulin sensitivity using the euglycemic insulin clamp technique suggested enhancement of insulin sensitivity by tglp-1 in volunteers with T1D (14), but this effect was modest and was not confirmed in another study of T1D (16). There is also evidence of effects of tglp-1 on non-insulin-mediated glucose disposal in subjects with type 2 diabetes (17) and on minimal-model-determined sensitivity to glucose in normal subjects (18); therefore, contributions of the metabolic effects of tglp-1 agonists that are independent of gastric emptying and that may affect meal-related glycemic excursions in T1D cannot be excluded. Improvement in glycemic control with intensive insulin therapy in T1D is limited by the risk of hypoglycemia and is associated with risk of excessive weight gain (19). The results of the present study and earlier studies (6 8) with tglp-1 agonists in T1D suggest that improvement in glycemic control with avoidance of hypoglycemia may be attainable by the use of these agents as congeners with insulin. With respect to weight gain, continuous sc infusion of tglp-1 through 6 wk in type 2 diabetes led to improvement in glycemic control accompanied by weight loss rather than weight gain (20). Thus, the reduction of food intake observed with administration of tglp-1 agonists in animals with diabetes (21) and in normal volunteers with dosage in the range under consideration (22) appears not to be overcome by release from the appetite suppressive effect of HPP (23) that

5 Dupré et al. Exendin-4 for Type 1 Diabetes J Clin Endocrinol Metab, July 2004, 89(7): TABLE 2. Paired studies of effects of exendin-4 administered sc 15 min before breakfast with usual insulin at time 0 min in eight volunteers with T1D with no endogenous insulin secretion compared with responses in six normal subjects Test GLU (mm) a HPP (pm) a GON (ng/liter) a Acet ( M) a FIRI (pm) Vehicle in T1D Exendin-4 in T1D Vehicle in normal subjects Time-averaged incremental AUCs for plasma GLU (0 240 min), HPP (0 240 min), GON (0 120 min), Acet (0 60 min), and FIRI (0 240 min). GLU, Glucose; GON, glucagon; Acet, acetaminophen; FIRI, free immunoreactive insulin. See Table 1 for conversion factors. a P 0.05 for vehicle vs. exendin-4. might result from inhibition of this hormone by tglp-1 agonists. We conclude that the potential benefits of treatment of insulin-requiring diabetes with tglp-1 agonist(s) warrant longer-term studies to evaluate the effects of tglp-1 agonists on metabolic control and nutritional status. Acknowledgments We are grateful for technical assistance from Christine Moogk, preparation of sterile exendin-4 solutions by Pamela Zabel, and nursing assistance from Janice McCallum and Margaret Watson. We are also grateful for operating support from Amylin Pharmaceuticals Inc., through an agreement of collaboration with London Health Sciences Research Inc. We thank our colleagues Dr. I. Hramiak, Dr. J. L. Mahon, and Dr. T. Paul for their continuing care of volunteers with type 1 diabetes during this study. Received November 17, Accepted April 6, Address all correspondence and requests for reprints to: John Dupré, Robarts Research Institute, P.O. Box 5015, 100 Perth Drive, London, Ontario, N6A 5K8 Canada. john.dupre@lhsc.on.ca. 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