Richard D. DiMarchi

A new glucagon and GLP-1 co-agonist eliminates obesity in rodents

We report the efficacy of a new peptide with agonism at the glucagon and GLP-1 receptors that has potent, sustained satiation-inducing and lipolytic effects. Selective chemical modification to glucagon resulted in a loss of specificity, with minimal change to inherent activity. The structural basis for the co-agonism appears to be a combination of local positional interactions and a change in secondary structure. Two co-agonist peptides differing from each other only in their level of glucagon receptor agonism were studied in rodent obesity models. Administration of PEGylated peptides once per week normalized adiposity and glucose tolerance in diet-induced obese mice. Reduction of body weight was achieved by a loss of body fat resulting from decreased food intake and increased energy expenditure. These preclinical studies indicate that when full GLP-1 agonism is augmented with an appropriate degree of glucagon receptor activation, body fat reduction can be substantially enhanced without any overt adverse effects.

Reduction of Postprandial Hyperglycemia and Frequency of Hypoglycemia in IDDM Patients on Insulin-Analog Treatment

Insulin lispro, an insulin analog recently developed particularly for mealtime therapy, has a fast absorption rate and a short duration of action. We compared insulin lispro and regular human insulin in the mealtime treatment of 1,008 patients with IDDM. The study was a 6-month randomized multinational (17 countries) and multicenter (102 investigators) clinical trial performed with an open-label crossover design. Insulin lispro was injected immediately before the meal, and regular human insulin was injected 30–45 min before the meal. Throughout the study, the postprandial rise in serum glucose was significantly lower during insulin lispro therapy. At the endpoint, the postprandial rise in serum glucose was reduced at 1 h by 1.3 mmol/l and at 2 h by 2.0 mmol/l in patients treated with insulin lispro (P < 0.001). The rate of hypoglycemia was 12% less with insulin lispro (6.4 ± 0.2 vs. 7.2 ± 0.3 episodes/30 days, P < 0.001), independent of basal insulin regimen or HbA1c level. The reduction was observed equally in episodes with and without symptoms. When the total number of episodes for each patient was analyzed according to the time of occurrence, the number of hypoglycemic episodes was less with insulin lispro than with regular human insulin therapy during three of four quarters of the day (P < 0.001). The largest relative improvement was observed at night. In conclusion, insulin lispro improves postprandial control, reduces hypoglycemic episodes, and improves patient convenience, compared with regular human insulin, in IDDM patients.

Unimolecular Dual Incretins Maximize Metabolic Benefits in Rodents, Monkeys, and Humans

Compared to best-in-class GLP-1 mono-agonists, unimolecular co-agonists of GLP-1 and GIP with optimized pharmacokinetics enhance glycemic and metabolic benefits in mammals. “Twincretins”: Two Is Better than One Despite obesity-linked diabetes approaching worldwide epidemic proportions and the growing recognition of it as a global health challenge, safe and effective medicines have remained largely elusive. Pharmacological options targeting multiple obesity and diabetes signaling pathways offer greater therapeutic potential compared to molecules targeting a single pathway. Finan et al. now report the discovery, characterization, and translational efficacy of a single molecule that acts equally on the receptors for the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). In rodent models of obesity and diabetes, this dual incretin co-agonist more effectively lowered body fat and corrected hyperglycemia than selective mono-agonists for the GLP-1 and GIP receptors. An enhanced insulinotropic effect translated from rodents to monkeys and humans, with substantially improved levels of glycosylated hemoglobin A1c (HbA1c) in humans with type 2 diabetes. The dual incretin was engineered with selective chemical modifications to enhance pharmacokinetics. This, in combination with its inherent mixed agonism, lowered the drug dose and ameliorated the dose-limiting nausea that has plagued selective GLP-1 therapies. These dual incretin co-agonists signify a new direction for unimolecular combination therapy and represent a new class of drug candidates for the treatment of metabolic diseases. We report the discovery and translational therapeutic efficacy of a peptide with potent, balanced co-agonism at both of the receptors for the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). This unimolecular dual incretin is derived from an intermixed sequence of GLP-1 and GIP, and demonstrated enhanced antihyperglycemic and insulinotropic efficacy relative to selective GLP-1 agonists. Notably, this superior efficacy translated across rodent models of obesity and diabetes, including db/db mice and ZDF rats, to primates (cynomolgus monkeys and humans). Furthermore, this co-agonist exhibited synergism in reducing fat mass in obese rodents, whereas a selective GIP agonist demonstrated negligible weight-lowering efficacy. The unimolecular dual incretins corrected two causal mechanisms of diabesity, adiposity-induced insulin resistance and pancreatic insulin deficiency, more effectively than did selective mono-agonists. The duration of action of the unimolecular dual incretins was refined through site-specific lipidation or PEGylation to support less frequent administration. These peptides provide comparable pharmacology to the native peptides and enhanced efficacy relative to similarly modified selective GLP-1 agonists. The pharmacokinetic enhancement lessened peak drug exposure and, in combination with less dependence on GLP-1–mediated pharmacology, avoided the adverse gastrointestinal effects that typify selective GLP-1–based agonists. This discovery and validation of a balanced and high-potency dual incretin agonist enables a more physiological approach to management of diseases associated with impaired glucose tolerance.

Leptin : Structure, function and biology

Leptin is an adipocyte-derived hormone that acts as a major regulator for food intake and energy homeostasis. Leptin deficiency or resistance can result in profound obesity, diabetes, and infertility in humans. Since its discovery, our understanding of leptins biological functions has expanded from anti-obesity to broad effects on reproduction, hematopoiesis, angiogenesis, blood pressure, bone mass, lymphoid organ homeostasis, and T lymphocyte systems. Leptin orchestrates complex biological effects through its receptors, expressed both centrally and peripherally. Leptin receptor belongs to the class I cytokine receptor superfamily. At least five isoforms of leptin receptor exist, primarily because of alternate splicing. The longest form is capable of full signal transduction. The short forms may serve as leptin binding proteins and play a role in leptin transporting across the blood-brain barrier. In this review, we present the crystal structure of leptin and the structural comparison with other four-helical cytokines, discuss the leptin-receptor binding models based on other cytokine-receptor complex structures, and summarize the most recent progress on leptin signal transduction pathways--especially its link to peripheral lipid metabolism through AMP-activated protein kinase and hepatic stearoyl-CoA desaturase-1 pathways. Furthermore, we propose the structure based design of leptin analogs with increased stability, improved potency, enhanced blood-brain barrier transport, and extended time action for future therapeutic application.

A rationally designed monomeric peptide triagonist corrects obesity and diabetes in rodents

We report the discovery of a new monomeric peptide that reduces body weight and diabetic complications in rodent models of obesity by acting as an agonist at three key metabolically-related peptide hormone receptors: glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon receptors. This triple agonist demonstrates supraphysiological potency and equally aligned constituent activities at each receptor, all without cross-reactivity at other related receptors. Such balanced unimolecular triple agonism proved superior to any existing dual coagonists and best-in-class monoagonists to reduce body weight, enhance glycemic control and reverse hepatic steatosis in relevant rodent models. Various loss-of-function models, including genetic knockout, pharmacological blockade and selective chemical knockout, confirmed contributions of each constituent activity in vivo. We demonstrate that these individual constituent activities harmonize to govern the overall metabolic efficacy, which predominantly results from synergistic glucagon action to increase energy expenditure, GLP-1 action to reduce caloric intake and improve glucose control, and GIP action to potentiate the incretin effect and buffer against the diabetogenic effect of inherent glucagon activity. These preclinical studies suggest that, so far, this unimolecular, polypharmaceutical strategy has potential to be the most effective pharmacological approach to reversing obesity and related metabolic disorders.

Targeted estrogen delivery reverses the metabolic syndrome

We report the development of a new combinatorial approach that allows for peptide-mediated selective tissue targeting of nuclear hormone pharmacology while eliminating adverse effects in other tissues. Specifically, we report the development of a glucagon-like peptide-1 (GLP-1)-estrogen conjugate that has superior sex-independent efficacy over either of the individual hormones alone to correct obesity, hyperglycemia and dyslipidemia in mice. The therapeutic benefits are driven by pleiotropic dual hormone action to improve energy, glucose and lipid metabolism, as shown by loss-of-function models and genetic action profiling. Notably, the peptide-based targeting strategy also prevents hallmark side effects of estrogen in male and female mice, such as reproductive endocrine toxicity and oncogenicity. Collectively, selective activation of estrogen receptors in GLP-1–targeted tissues produces unprecedented efficacy to enhance the metabolic benefits of GLP-1 agonism. This example of targeting the metabolic syndrome represents the discovery of a new class of therapeutics that enables synergistic co-agonism through peptide-based selective delivery of small molecules. Although our observations with the GLP-1–estrogen conjugate justify translational studies for diabetes and obesity, the multitude of other possible combinations of peptides and small molecules may offer equal promise for other diseases.

Ghrelin promotes and protects nigrostriatal dopamine function via a UCP2-dependent mitochondrial mechanism.

Ghrelin targets the hypothalamus to regulate food intake and adiposity. Endogenous ghrelin receptors [growth hormone secretagogue receptor (GHSR)] are also present in extrahypothalamic sites where they promote circuit activity associated with learning and memory, and reward seeking behavior. Here, we show that the substantia nigra pars compacta (SNpc), a brain region where dopamine (DA) cell degeneration leads to Parkinsons disease (PD), expresses GHSR. Ghrelin binds to SNpc cells, electrically activates SNpc DA neurons, increases tyrosine hydroxylase mRNA and increases DA concentration in the dorsal striatum. Exogenous ghrelin administration decreased SNpc DA cell loss and restricted striatal dopamine loss after 1-methyl-4-phenyl-1,2,5,6 tetrahydropyridine (MPTP) treatment. Genetic ablation of ghrelin or the ghrelin receptor (GHSR) increased SNpc DA cell loss and lowered striatal dopamine levels after MPTP treatment, an effect that was reversed by selective reactivation of GHSR in catecholaminergic neurons. Ghrelin-induced neuroprotection was dependent on the mitochondrial redox state via uncoupling protein 2 (UCP2)-dependent alterations in mitochondrial respiration, reactive oxygen species production, and biogenesis. Together, our data reveal that peripheral ghrelin plays an important role in the maintenance and protection of normal nigrostriatal dopamine function by activating UCP2-dependent mitochondrial mechanisms. These studies support ghrelin as a novel therapeutic strategy to combat neurodegeneration, loss of appetite and body weight associated with PD. Finally, we discuss the potential implications of these studies on the link between obesity and neurodegeneration.

The metabolic actions of glucagon revisited

The initial identification of glucagon as a counter-regulatory hormone to insulin revealed this hormone to be of largely singular physiological and pharmacological purpose. Glucagon agonism, however, has also been shown to exert effects on lipid metabolism, energy balance, body adipose tissue mass and food intake. The ability of glucagon to stimulate energy expenditure, along with its hypolipidemic and satiating effects, in particular, make this hormone an attractive pharmaceutical agent for the treatment of dyslipidemia and obesity. Studies that describe novel preclinical applications of glucagon, alone and in concert with glucagon-like peptide 1 agonism, have revealed potential benefits of glucagon agonism in the treatment of the metabolic syndrome. Collectively, these observations challenge us to thoroughly investigate the physiology and therapeutic potential of insulins long-known opponent.

Synthesis and Conformational Analysis of a Cyclic Peptide Obtained via i to i+4 Intramolecular Side-Chain to Side-Chain Azide-Alkyne 1,3-Dipolar Cycloaddition

Intramolecular side-chain to side-chain cyclization is an established approach to achieve stabilization of specific conformations and a recognized strategy to improve resistance toward proteolytic degradation. To this end, cyclizations, which are bioisosteric to the lactam-type side-chain to side-chain modification and do not require orthogonal protection schemes, are of great interest. Herein, we report the employment of Cu(I)-catalyzed 1,3-dipolar cycloaddition of side chains modified with azido and alkynyl functions and explore alternative synthetic routes to efficiently generate 1,4-disubstituted [1,2,3]triazolyl-containing cyclopeptides. The solid-phase assembly of the linear precursor including epsilon-azido norleucine and the propargylglycine (Pra) in positions i and i+4, respectively, was accomplished by either subjecting the resin-bound peptide to selective on-resin diazo transformation of a Lys into the Nle(epsilon-N3) or the incorporation of Fmoc-Nle(epsilon-N3)-OH during the stepwise build-up of the resin-bound peptide 1b. Solution-phase Cu(I)-catalyzed 1,3-dipolar cycloaddition converts the linear precursor Ac-Lys-Gly-Nle(epsilon-N3)-Ser-Ile-Gln-Pra-Leu-Arg-NH2 (2) into the 1,4-disubstituted [1,2,3]triazolyl-containing cyclopeptide [Ac-Lys-Gly-Xaa(&(1))-Ser-Ile-Gln-Yaa(&(2))-Leu-Arg-NH2][(&(1)(CH2)4-1,4-[1,2,3]triazolyl-CH2&(2))] (3). The conformational preferences of the model cyclopeptide 3 (III), which is derived from the sequence of a highly helical and potent i to i+4 side-chain to side-chain lactam-containing antagonist of parathyroid hormone-related peptide (PTHrP), are compared to the corresponding lactam analogue Ac[Lys(13)(&(1)),Asp(17)(&(2))]hPTHrP(11-19)NH2 (II). CD and NMR studies of 3 and II in water/hexafluoroacetone (HFA) (50:50, v/v) revealed a high prevalence of turn-helical structures involving in particular the cyclic regions of the molecule. Despite a slight difference of the backbone arrangement, the side-chains of Ser, Gln, and Ile located at the i+1 to i+3 of the ring-forming sequences share the same spatial orientation. Both cyclopeptides differ regarding the location of the turn-helical segment, which in II involves noncyclized residues while in 3 it overlaps with residues involved in the cyclic structure. Therefore, the synthetic accessibility and conformational similarity of i to i+4 side-chain to side-chain cyclopeptide containing the 1,4-disubstituted [1,2,3]triazolyl moiety to the lactam-type one may result in similar bioactivities.

Modifications in the B10 and B26–30 regions of the B chain of human insulin alter affinity for the human IGF-I receptor more than for the insulin receptor

Summary Inversion of the natural sequence of the B chain of human insulin (HI) from ProB28LysB29 to LysB28ProB29 generates an insulin analogue with reduced tendency to self-associate. Since this substitution increases the homology of insulin to insulin-like growth factor-I (IGF-I), we have examined the affinity of a series of insulin analogues with the general modified structure XaaB28ProB29 HI for binding to both human placental insulin and IGF-I receptors. The XaaB28ProB29 HI series is approximately equipotent to HI in binding to the insulin receptor with the exception of when Xaa = Phe, Trp, Leu, Ile, and Gly (40–60 % relative to HI). Substitution with basic residues in the B28 position increased the relative affinity to the IGF-I receptor approximately 1.5−2-fold (ArgB28ProB29 > OrnB28ProB29 = LysB28ProB29). Substitution with acidic residues reduced relative affinity for the IGF-I receptor approximately 2-fold (CyaB28ProB29 = GluB28ProB29 > AspB28ProB29). Combination of AspB10 substitution in conjunction with a modification in the B28–29 position (e.g. AspB10LysB28ProB29 HI) showed an additional 2-fold selective increase in affinity for the IGF-I receptor, suggesting that these two effects are additive. Addition of Arg residues at B31–32, on the backbone of either HI or AspB10 HI, increased affinity for the IGF-I receptor 10 and 28 fold, respectively, compared to HI, confirming the significance of enhanced positive charge at the C-terminal end of the insulin B-chain in increasing selectivity for the IGF-I receptor. This relative increase in IGF-I receptor affinity correlated largely, but not completely, with enhanced growth promoting activity in human mammary epithelial cells. In the case of LysB28ProB29 HI, growth activity correlated with dissociation kinetics from the insulin receptor which were shown to be identical with those of human insulin. [Diabetologia (1997) 40: S 54–S 61]