Amylin Pharmaceuticals, Inc., 9360 Towne Centre Drive, San Diego, CA 92121. E-mail: email@example.com
Objective: We previously reported that a single preprandial injection (120 μg) of pramlintide, an analog of the β-cell hormone amylin, reduced ad libitum food intake in obese subjects. To further characterize the meal-related effects of amylin signaling in humans, we studied a lower pramlintide dose (30 μg) in normal-weight subjects.
Research Methods and Procedures: In a randomized, double-blind, placebo-controlled, cross-over study, 15 healthy men (age, 24 ± 7 years; BMI, 22.2 ± 1.8 kg/m2) underwent a standardized buffet meal test on two occasions. After an overnight fast, subjects received a single subcutaneous injection of pramlintide (30 μg) or placebo, followed immediately by a standardized pre-load meal. After 1 hour, subjects were offered an ad libitum buffet meal, and total caloric intake and meal duration were measured.
Results: Compared with placebo, pramlintide reduced total caloric intake (1411 ± 94 vs. 1190 ± 117 kcal; Δ, −221 ± 101 kcal; −14 ± 9%; p = 0.05) and meal duration (36 ± 2 vs. 31 ± 3 minutes; Δ, −5.1 ± 1.4 minutes; p < 0.005). Visual analog scale profiles of hunger trended lower and fullness higher during the first hour after pramlintide administration. In response to the buffet, hunger and fullness changed to a similar degree after pramlintide and placebo, despite subjects on pramlintide consuming 14% fewer kilocalories. Visual analog scale nausea ratings remained near baseline, without differences between treatments. Plasma peptide YY, cholecystokinin, and ghrelin concentrations did not differ with treatment, whereas glucagon-like peptide-1 concentrations after meals were lower in response to pramlintide than to placebo.
Discussion: These observations add support to the concept that amylin agonism may have a role in human appetite control.
In recent years, the discovery and characterization of novel gastrointestinal peptide hormones, such as ghrelin and peptide YY-3–36 (PYY3–36),1 have sparked renewed interest concerning the physiologic role of peripheral satiety signals in the complex neuroendocrine control of food intake and body weight (1)(2)(3)(4)(5). Specifically, there is growing recognition that peptide hormones secreted from the gut and pancreatic islets may exert orexigenic [ghrelin (6)] and anorexigenic [e.g., cholecystokinin (CCK) (7)(8), glucagon-like peptide-1 (GLP-1) (9)(10)(11), PYY3–36 (12)(13), and pancreatic polypeptide (14)] effects that may contribute to the short-term control of food intake by regulating the initiation and/or termination of meals.
Interest in the role of pancreatic β cells in energy homeostasis has thus far focused on the potential role of insulin in long-term body weight regulation. The fact that circulating insulin concentrations increase in proportion to body fat, coupled with recent advances in our understanding of hypothalamic insulin signaling (15)(16), support a role for insulin as a long-term adiposity signal. Because β-cell hormone secretion is pulsatile in nature and tightly linked to the ingestion of nutrients and their subsequent appearance in the circulation, it is conceivable that β cell–derived signals may also be involved in the short-term regulation of food intake.
Amylin is a 37 amino acid peptide hormone co-secreted by pancreatic β cells in conjunction with insulin (17). In healthy subjects, plasma amylin concentrations rise rapidly and by several-fold in response to meals, with a diurnal profile that is almost superimposable on that of insulin (17). On secretion into the circulation, amylin binds with high affinity to specific amylin receptors in the central nervous system, thus acting as a neuroendocrine hormone. A major binding site for amylin is the area postrema, which, along with the adjacent nucleus of the solitary tract, lacks a blood–brain barrier for diffusion of large molecules (18)(19)(20). This region of the hindbrain serves an important role in the reception and integration of peripheral (humoral and vagal afferent) satiety signals (1)(5).
Several non-clinical studies have reported that peripheral administration of amylin dose-dependently reduces food intake and meal size in rodents (21)(22)(23)(24)(25), with anorexigenic activity ensuing at doses as low as 10 μg/kg (26). Moreover, attenuation of endogenous amylin signaling through central or peripheral administration of a selective amylin antagonist has been shown to increase food intake and meal frequency in rats (27)(28), supporting the notion that amylin signaling may have a physiologic role in appetite regulation.
Our recent results from a randomized, double-blind, placebo-controlled, cross-over study showed that the amylin analog pramlintide, at a dose of 120 μg subcutaneously, reduced ad libitum food intake at a buffet meal and enhanced meal-related satiation in obese subjects and subjects with type 2 diabetes (29).
To further characterize the meal-related effects of amylin signaling in humans, we studied an additional group of healthy, normal-weight subjects, using the same experimental buffet-meal setting but with a substantially lower dose of pramlintide (30 μg). To assess whether the satiogenic effect of amylin agonism is primary or secondary in nature, we also measured circulating concentrations of several gastrointestinal hormones known to have stimulatory or inhibitory effects on food intake.
Research Methods and Procedures
A total of 15 healthy, normal-weight male subjects were studied. Because of the potential confounding effect of the menstrual cycle on hunger and food intake (30), women were excluded from the study.
Primary inclusion criteria required that subjects were between 18 and 70 years of age, with a baseline BMI of ≥20 and ≤25 kg/m2, had pre-experimental weight fluctuations of <5%, had normal thyroid stimulating hormone concentrations, and were unrestrained eaters [score <11 on Factor 1 (cognitive restraint) based on the Three-Factor Eating Questionnaire (31)]. Primary exclusion criteria included, but were not limited to, cardiac disease, uncontrolled hypertension, gastrointestinal, hepatic, renal, autoimmune, or hematological disease, eating disorders, or current treatment with systemic steroids, anti-obesity agents, anti-depressants, anti-psychotic drugs, or drugs known to affect gastrointestinal motility.
The study was designed in accordance with the Declaration of Helsinki and approved by the Royal Adelaide Hospital Human Ethics Committee. All participants provided informed consent before randomization.
This was a single-center, randomized, double-blind, placebo-controlled, two-period, cross-over study. Subjects were instructed to fast from 10:00 pm the previous night until they reported to the clinical research unit the following morning. A minimum of 72 hours was required between each study visit, during which subjects were asked to maintain their usual dietary habits. Identical standardized meals were served on both study visits.
At t = −60 minutes, vital signs and weight were measured, and subjects were fitted (antecubital vein) with a cannula for blood sampling. Blood samples and visual analog scale (VAS) assessments were taken at similar time-points for 5.5 hours.
At t = 0 minutes, subjects received, in randomized order, a single subcutaneous injection of 30 μg pramlintide or placebo (Amylin Pharmaceuticals, Inc., San Diego, CA). Immediately after the injection, subjects consumed within 3 minutes a standardized pre-load meal consisting of 125 grams banana blended with 150 mL low-fat (2%) milk and 150 mL water (estimated energy content: ∼189 kcal; 6 grams protein, 36 grams carbohydrate, 3 grams fat).
One hour later (t = 60 minutes), an ad libitum buffet meal was offered to the subjects for 45 minutes (t = 60 to 105 minutes). The buffet selections included ham and chicken sandwiches prepared on white or whole wheat bread with a variety of condiments/toppings (cheese, tomato, lettuce, cucumber, butter, and mayonnaise), as well as yogurt, fruit salad, individual fruits, custard, orange juice, and coffee. Food availability exceeded the predicted intake.
Total caloric and macronutrient intakes were calculated using the DIET/4 program (9) (Xyris Software, Highgate Hill, Queensland, Australia). Meal duration (i.e., total duration of eating at the ad libitum buffet meal) was also recorded. Subjects were asked to rate sensations of hunger, fullness, and nausea over the course of the meal test using a paper-based standardized 100-mm VAS (8)(32). Blood samples were collected at regular intervals to measure postprandial hormonal responses, including plasma concentrations of ghrelin [radioimmunoassay assay (RIA) intra- and inter-assay precision is ≤10% and 15%, respectively] (33), CCK (RIA intra- and inter-assay precision of ≤9% and 27%, respectively) (34)(35), total GLP-1 (RIA intra- and inter-assay precision of ≤15% and 18%, respectively), and total PYY (RIA intra- and inter-assay precision of ≤10% and 9%, respectively) (33). Ghrelin, total GLP-1, and total PYY assays were run by Linco Research, Inc. (St. Charles, MO).
The intent-to-treat population was used for all analyses. Total caloric intake, macronutrient intake, and meal duration were summarized descriptively by treatment. Incremental VAS hunger, fullness, and nausea score profiles, as well as incremental ghrelin, CCK, GLP-1, and PYY concentration time profiles, were calculated by subtracting the baseline score (collected at t = 0 minutes) from the score at each subsequent time-point. The linear trapezoidal method was used to calculate incremental areas under the percentage score/concentration-time curve from 0 to 1 (premeal period), 1 to 5 (peri- and post-meal period), and 0 to 5 hours (entire period).
Data are presented as mean ± SE for all parameters. Pramlintide and placebo results were analyzed using mixed-effect models that included treatment, treatment sequence, and period as fixed effects and subject-within-sequence as a random effect. From the mixed-effect models, the least squares (LS) means, LS mean differences between pramlintide and placebo, corresponding SEs, 95% confidence intervals, and p values for the LS mean differences were derived.
In addition, correlation analyses were used to study the inter-relationships among total caloric intake, meal duration, and concomitant VAS ratings of hunger and fullness. The Pearson correlation coefficients (r with p values) are presented; p < 0.05 was considered significant.
The 15 healthy, normal-weight male subjects had a mean age of 24 ± 7 years (range: 18 to 45 years), a mean weight of 71.6 ± 7.5 kg (range: 56.5 to 82.0 kg), and a mean BMI of 22.2 ± 1.8 kg/m2 (range: 19.1 to 25.1 kg/m2). All 15 randomized subjects completed the study and were considered evaluable.
Food Intake and Meal Duration
Total caloric intake at the buffet meal was reduced by 221 ± 101 kcal (p = 0.05), or 14%, after pramlintide (1190 ± 117 kcal) compared with placebo administration (1411 ± 94 kcal; Figure 1A).
Meal duration was reduced 5.1 ± 1.4 minutes (p < 0.005), or 14%, after pramlintide (30.7 ± 2.6 minutes) compared with placebo (35.8 ± 2.2 minutes; Figure 1B).
Correlation analyses revealed a significant positive relationship between meal duration and total caloric intake whether subjects were treated with pramlintide (r = 0.674, p < 0.01) or placebo (r = 0.630, p < 0.05; Figure 2A).
VAS Ratings of Hunger, Fullness, and Nausea
Hunger and Fullness
With both treatments, subjects experienced a decrease in mean hunger ratings in response to the pre-load meal (0 to 1 hour) and, to a greater extent, after the ad libitum buffet (1 to 1.75 hours; Figure 3A). During the postprandial period, hunger ratings increased but had not yet returned to baseline by 5 hours. Reciprocal changes were seen with mean fullness ratings.
After the pre-load (0 to 1 hour), the decrease in hunger ratings and increase in fullness ratings tended to be more marked after pramlintide than after placebo administration, although the differences in the incremental area under the curve 0–1 hour were not statistically significant (Figure 3Aand 3B). Consequently, at the beginning of the buffet meal (t = 1 hour), the mean hunger rating trended lower and the mean fullness rating trended higher for pramlintide compared with placebo administration. Correlation analysis revealed a positive correlation between the premeal hunger rating and subsequent caloric intake (Figure 2B). Immediately after the buffet meal, the hunger and fullness responses were comparable for both treatments, despite the fact that total caloric intake at the buffet meal was 14% lower after pramlintide (Figure 3A).
There were no discernible differences between pramlintide and placebo treatment relative to mean nausea ratings, which changed only minimally during the course of the experiments (Figure 3C).
Postprandial Hormonal Analytes
Plasma concentrations of ghrelin decreased slightly after the pre-load and decreased more markedly after the buffet meal. During the postprandial period, plasma ghrelin concentrations increased but had not yet returned to baseline by 5 hours (Figure 4A). The ghrelin profile in response to the pre-load and during the postprandial period was similar after pramlintide and placebo administration. After the buffet meal (1.75 hours), the incremental suppression of ghrelin appeared greater with pramlintide compared with placebo (Δ,−159 vs. −96 pg/mL), despite subjects eating less on the pramlintide day, but this difference was not statistically significant.
CCK, GLP-1 (Total), and PYY (Total)
The plasma concentration of CCK, GLP-1, and PYY increased after the pre-load, increased more markedly during the buffet, and decreased during the postprandial period, with none of the analytes returning to baseline by 5 hours (Figure 4).
The CCK profile was similar following pramlintide and placebo administration.
The total GLP-1 response to the pre-load and buffet meals appeared attenuated after pramlintide treatment. Only the incremental area under the curve0–1 hour after the pre-load meal was significantly different for pramlintide compared with placebo [−0.40 ± 0.4 vs. 1.9 ± 0.4 pM; p < 0.005].
The total PYY response to the meals also seemed attenuated after pramlintide compared with placebo, although the differences in areas under the curve were not statistically significant.
Pramlintide generally was well tolerated, with no serious adverse events occurring. The only treatment emergent adverse event was one subject reporting moderate nausea that developed ∼5 hours after dosing, and it was deemed unrelated to pramlintide administration.
In a detailed review of the non-clinical literature, Lutz (36) suggested that amylin fulfills all five criteria of a physiologic satiety signal: meal-induced secretion of the peptide, rapid onset of activity, dose-dependent anorexigenic activity, the absence of adverse effects, and activity at doses similar to physiologic concentrations (although the latter point was inferred in large part from receptor antagonist studies). Because of the difficulties of inferring satiety effects from rodent studies, this study is an important contribution in that it provides clinical evidence with an amylin agonist at near-physiologic concentrations, supporting the concept that amylin agonism may play a physiologic role in the regulation of food intake, meal termination, and satiety in humans.
Because of low solubility and a tendency for self-aggregation, human amylin itself cannot be readily administered to humans. For this reason, pramlintide was developed by substituting proline for three amino acids in the amylin sequence (Ala 25, Ser 28, Ser 29). The resulting peptide, pramlintide, is stable, soluble, and non-aggregating. Importantly, the broad range of pharmacologic actions of pramlintide, including receptor binding, is preserved and virtually indistinguishable from amylin (37). Thus, in the interpretation of this study, amylin and pramlintide can be considered equipotent.
This study established that, in healthy, normal-weight subjects, a single subcutaneous 30-μg injection of pramlintide significantly reduced total caloric intake by an average of 221 kcal (14%) during an ad libitum buffet meal. The reduction in total caloric intake was similar in magnitude to the reduction previously observed with a 120-μg dose of pramlintide in obese subjects, as well as insulin-treated patients with type 2 diabetes (29). Notwithstanding the methodological limitations in establishing the physiologic role of peripheral satiety signals in humans, our results with low-dose pramlintide provide supporting evidence that amylin agonism may contribute to human appetite control. The secretion profile of endogenous amylin in response to mixed meals and an oral glucose tolerance test has been previously measured (38)(39)(40)(41). Using a monoclonal antibody-based, two-site enzyme linked immunosorbent assay to measure intact amylin, plasma concentrations of endogenous amylin were found to increase several-fold during the postprandial period, to ∼20 pM in healthy individuals and 45 pM in obese individuals with impaired glucose tolerance, with a peak at ∼30 minutes after a meal, followed by a progressive decline toward fasting levels within 2 to 3 hours (38). Although the pharmacokinetic profile of pramlintide was not measured in this study, it is known from previous studies that plasma pramlintide concentrations after a single subcutaneous injection of 30 μg peak at ∼38 pM and decline to <20 pM within 1 hour after injection (17)(42). It can, therefore, be assumed that the prevailing plasma pramlintide concentration before and during the ad libitum buffet meal was within or near the range of physiologic amylin responses to a mixed meal. This finding is generally consistent with the anorexigenic threshold doses established in carefully conducted rodent studies, which reported that near physiologic concentrations of amylin (an increase of ∼10 or ∼24 pM achieved through continuous infusion) induced a 26% and 34% reduction in 2-hour food intake in rats (43)(44).
Further analyses of our data revealed that total caloric intake and meal duration were significantly correlated after both placebo and pramlintide administration. Although the physiologic basis of the relationship remains to be elucidated, the results are generally consistent with a role of amylin signaling in the sequence of peripherally and centrally mediated events that determine meal size and termination.
Having established that food intake was reduced after pramlintide treatment, the concomitant changes in subjective ratings of hunger, fullness, and nausea were examined. The perceptions of enhanced preprandial fullness and hunger suppression during the first hour after pramlintide administration support the concept that the reduction in food intake was attributable to a satiogenic effect. This was further supported by the finding that hunger ratings at the initiation of the buffet meal were predictive of subsequent food intake and that changes in hunger and fullness during and after the buffet meal were similar on both occasions, despite a significantly lower caloric intake with pramlintide. While gastric emptying was not measured in this study, it is conceivable that the effect of pramlintide on fullness could, at least in part, be related to the peptide's well-known effect to slow gastric emptying (17). Specifically, results from a recent study with CCK and GLP-1 have implicated pyloric motility as a potential contributor to the effect of anorexigenic gut peptides on food intake and appetite regulation (45).
The observed reduction in food intake by pramlintide was independent of nausea, since mean VAS nausea ratings remained unchanged during the course of the experiments and were indistinguishable between pramlintide and placebo. Because there were also no reports of treatment-related nausea during the 5-hour study period of either day, it is unlikely that our findings were confounded by lack of tolerability.
Consistent with our previous observations in obese subjects and obese insulin-treated subjects with type 2 diabetes (29), this study provided no evidence that the effect of pramlintide to reduce food intake is mediated by other anorexigenic peptide hormones. On the contrary, pramlintide significantly attenuated the response of GLP-1 to the pre-load meal and seemed to have similar, albeit non-significant, effects on CCK and total PYY. GLP-1 and total PYY concentrations may have increased less after the buffet meal following pramlintide compared with placebo injection because of the pramlintide-induced reduction in food intake. However, this would not explain the apparent lack of effect on postprandial CCK concentrations.
An interesting finding following pramlintide administration was the observed trend toward a greater periprandial suppression of circulating ghrelin, a gastric hormone with putative orexigenic effects. Although ghrelin is commonly thought to have its primary physiologic role in meal initiation (46), it cannot be excluded that the greater fall in ghrelin during the buffet meal may have contributed to the reductions in food intake and meal duration following pramlintide injection. In fact, this would be consistent with studies in rodents, showing that plasma concentrations of both total and active ghrelin are potently and dose-dependently suppressed by amylin and enhanced by the selective amylin antagonist AC187 (47). Although it cannot be determined from the observational data in this study whether ghrelin suppression may have contributed to the reduction in food intake induced by pramlintide, our results are consistent with the notion that the β-cell hormone amylin may be a physiologic inhibitor of ghrelin secretion (47).
In summary, compared with placebo, a low (30 μg) dose, single subcutaneous injection of pramlintide elicited significant reductions in food intake and meal duration in healthy, normal-weight subjects. These effects seem to be brought about by a primary satiogenic effect, are clearly dissociated from the occurrence of nausea, and are unlikely to be mediated by other anorexigenic gut peptides. These observations with pramlintide add support to the concept that amylin agonism may have a role in meal-related satiation.
We thank Sharon Skare, Derek Deckhut, Shereen McIntyre, Suzann Hahs, and Lori Nowland for excellent assistance in the conduct, reporting, and quality control of the study and Nicole Kesty for manuscript preparation. This work was supported by Amylin Pharmaceuticals, Inc.
Nonstandard abbreviations: PYY, peptide YY; CCK, cholecystokinin; GLP-1, glucagon-like peptide-1; VAS, visual analog scale; RIA, radioimmunoassay; LS, least squares.
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