A Peptidomimetic Targeting White Fat Causes Weight Loss and Improved Insulin Resistance in Obese Monkeys
Obesity, defined as body mass index greater than 30, is a leading cause of morbidity and mortality and a financial burden worldwide. Despite significant efforts in the past decade, very few drugs have been successfully developed for the treatment of obese patients. Biological differences between rodents and primates are a major hurdle for translation of anti-obesity strategies either discovered or developed in rodents into effective human therapeutics. Here, we evaluate the ligand-directed peptidomimetic CKGGRAKDC-GG-D(KLAKLAK)2 (henceforth termed adipotide) in obese Old World monkeys. Treatment with adipotide induced targeted apoptosis within blood vessels of white adipose tissue and resulted in rapid weight loss and improved insulin resistance in obese monkeys. Magnetic resonance imaging and dual-energy x-ray absorptiometry confirmed a marked reduction in white adipose tissue. At experimentally determined optimal doses, monkeys from three different species displayed predictable and reversible changes in renal proximal tubule function. Together, these data in primates establish adipotide as a prototype in a new class of candidate drugs that may be useful for treating obesity in humans.
INTRODUCTION
Unless current trends are reversed, the epidemic of human obesity and its associated comorbidities will account for a major fraction of healthcare costs worldwide (1, 2). An international collaborative prospective analysis of nearly 1 million participants concluded that obesity is associated with increased overall and cause-specific mortality in a magnitude roughly equivalent to that of smoking (3). Given that programs to promote life-style changes in diet and exercise have been insufficient to address this chronic human condition, aggressive research of anti-obesity compounds has been pursued by the pharmaceutical and biotechnology industries. However, effective drugs have proven extremely difficult to develop (4). Currently, only two Food and Drug Administration (FDA)–approved drugs for weight loss are available in the United States: the appetite suppressant phentermine and the inhibitor of fat absorption orlistat. Despite the initial popularity of these drugs, placebo-subtracted weight losses are actually quite limited, and concerns over side effects continue to limit their use (5–8).
Conventional pharmacologic treatment of obesity relies on central nervous system (CNS) and/or peripheral metabolic mechanisms to suppress appetite and elevate energy expenditure (6, 9, 10). Many of the recently developed candidate anti-obesity drugs are centrally acting and have been associated with serious adverse events that include unanticipated cardiovascular, pulmonary, and neuropsychiatric toxicity. Moreover, sibutramine has been withdrawn from the U.S. market (8, 11, 12), and the FDA has withheld the approval of three highly anticipated anti-obesity agents because of various safety concerns. Given this conservative approach of regulatory agencies to approving pharmaceutical agents directed at the CNS, it is increasingly clear that new approaches for inducing weight loss will be essential for developing drugs to successfully treat human obesity.
In pivotal work, Rupnick and colleagues demonstrated that adipose tissue mass can be regulated through the vasculature in obese mice (13). These findings provided a conceptual framework for the use of angiogenesis inhibitors as drug candidates for weight loss. Subsequently, in mouse models, functional roles for progenitor cells of white adipose tissue within the vascular niche have been found in normal fat tissue (14) and experimental tumors (15), suggesting a cellular contribution to angiogenesis in obesity.
In previous work, our group and others reported obesity reversal by targeted induction of apoptosis in blood vessels supplying white adipose tissue in obese mice (16) and rats (17). A combinatorial phage display random peptide library selection in vivo yielded a cyclic motif (sequence CKGGRAKDC) that selectively targets endothelial cell surface expression of the receptor prohibitin within the vasculature of white adipose tissue (16). This prohibitin-binding motif was chemically fused to the d-enantiomer D(KLAKLAK)2 sequence, an amphipathic peptidomimetic that disrupts mitochondrial membranes upon receptor-mediated cell internalization and causes targeted apoptosis (16–23). Specifically, the peptidomimetic CKGGRAKDC-GG-D(KLAKLAK)2, hereafter referred to as adipotide, targeted the vasculature of white adipose tissue and resulted in ~30% weight reduction in obese mice over a period of 4 weeks (16). Therefore, unlike nonspecific angiogenesis inhibitors, adipotide is systemically targeted to the endothelium of fat through a ligand-directed mechanism and disrupts the vascular supply of white adipose tissue, at least in rodent obesity models (16, 17). Moreover, an annexin A2–prohibitin receptor system targeted by adipotide has been reported recently in the white adipose tissue vasculature of human patients (24).
The failure of standard research in rodents to model human obesity has been a major hurdle to the discovery, development, and approval of new anti-obesity compounds. Indeed, biological differences limit the relevance of data derived from rodent studies that can be translated into drugs against human obesity (25). Particularly problematic research areas include the differential functions of certain adipokines (26) and peptide hormones (27), responses to neurotransmitter stimuli (28), and differences in circadian rhythms and/or feeding behaviors (29) between rodents and primates, including humans.
To address these challenges, we evaluated whether the marked response to adipotide observed in rodent models of obesity (16, 17) could be recapitulated in obese nonhuman primates. In contrast to other mammalian species (for example, mice, rats, rabbits, and dogs), humans and Old World monkeys share considerable physiological features such as the metabolic response to lipolysis in white fat adipocytes (28) and pathological obesity-related conditions such as the development of cardiovascular disease and insulin resistance or even overt type 2 diabetes mellitus (30). Here, we analyzed three species of Old World monkeys: rhesus macaques, baboons, and cynomolgus macaques.
RESULTS
Therapeutic dose-finding study of adipotide in a nonhuman primate model of obesity
We first used spontaneously obese rhesus macaques (Macaca mulatta), one of the most relevant primate models for human obesity and its associated comorbidities. In an initial dose-finding study, female rhesus macaques (n = 4) aged 9 to 13 years were selected for study entry on the basis of a history of spontaneous obesity, increased food consumption, and failure to maintain a high level of activity. All animals were defined as obese by a body mass index (BMI) ranging from 34 to 45 according to the equivalent formula for rhesus macaques (31). Body weights ranged from 10.0 to 11.7 kg, and the heaviest monkey had a morbidly obese body habitus (BMI = 45). All monkeys had the most clinically abundant abdominal fat in a central pattern relative to that of a large cohort of adult females (n = 350) in the breeding colony. Individual monkeys were euglycemic, with fasting serum glucose levels ranging from 65 to 71 mg/dl (normal range, <80 mg/dl) and serum insulin levels ranging from 21 to 52 μU/ml (normal range, <41 μU/ml). Adipotide was prepared under Good Manufacturing Practice (GMP) to our specifications by PolyPeptide Laboratories. Daily subcutaneous treatment with increasing doses of adipotide resulted in a dose-dependent decrease in body weight, BMI, and abdominal circumference relative to that of negative control monkeys receiving saline (Fig. 1, A to C). After dosing for 9 weeks, the body weight of the two treated monkeys in this initial cohort decreased by 1.8 kg (15.4%) and 2.1 kg (20.4%; Fig. 1A). Moreover, the BMI decreased from 45.0 to 37.3 (17.1%) in the morbidly obese monkey and from 38.8 to 30.9 (20.4%) in the second treated monkey (Fig. 1B). Finally, abdominal circumferences were reduced by 6.5 cm (13.1%) and 9.0 cm (14.2%) from baseline (pretreatment) measurements (Fig. 1C). During the same time period, the body weight, BMI, and abdominal circumference of the obese control monkeys receiving only saline did not change.
At the baseline evaluation, the morbidly obese monkey was deemed insulin-resistant on the basis of an intravenous glucose tolerance test (IVGTT); 60 min after the initial administration of glucose, the serum glucose level remained mildly elevated at 100 mg/dl (fig. S1A). The other three obese monkeys displayed relatively normal serum insulin responses to glucose administration (fig. S1, C, E, and G). A second IVGTT was performed 3 days after the final administration of adipotide. The area under the curve (AUC) for insulin decreased 61.4% and 63.5% in the two monkeys treated with adipotide (fig. S1 and table S1). These findings indicated a considerable decrease in insulin resistance based on the reduced level of insulin required to respond to acute doses of intravenous glucose.
Efficacy of adipotide at the therapeutic dose in a nonhuman primate model of obesity
Based on the dose-finding study, we determined the optimal subcutaneous dose of adipotide (0.43 mg/kg). Subsequently, we performed a second study on a larger cohort (n = 15 obese rhesus macaques; 5 controls and 10 treated) to evaluate the effect of the optimal fixed dose of adipotide daily for 4 weeks, followed by a 4-week recovery period. At the end of the treatment interval, the weight of the control monkeys had changed from +1.0 to −3.5% from pretreatment body weight, whereas the monkeys receiving the therapy displayed marked weight loss that ranged from −7.4 to −14.7% of pretreatment body weight (Fig. 2A). The BMI of the monkeys in the treatment group decreased at the end of treatment compared to pretreatment by −3.7 to −17.3%, whereas the control group exhibited a −3.5 to +3.3% BMI change (Fig. 2B). At the end of the treatment period, the abdominal circumference had decreased by −6.5 to −14.3% in 9 of 10 monkeys in the treated group; a single monkey displayed a 2% increase in abdominal circumference. In comparison, the change in abdominal circumference of the control group ranged from −6.3 to +6.9% (Fig. 2C). Body weight, BMI, and abdominal circumference continued to decrease for an additional 3 weeks after cessation of adipotide treatment. The change in these three variables over time from the start of treatment through the end of the recovery period was statistically significant [mixed-effects model (32), P < 0.0001 for each variable] between the treated and the control groups (Fig. 2, D to F).
