Patricia Grasso

In vivo effects of leptin-related synthetic peptides on body weight and food intake in female ob/ob mice: localization of leptin activity to domains between amino acid residues 106-140.

In C57BL/6J ob/ob mice, a single base mutation of the ob gene in codon 105 results in the replacement of arginine by a premature stop codon and production of a truncated inactive form of leptin. These observations suggest that leptin activity may be localized, at least in part, to domains distal to amino acid residue 104. To investigate this possibility, we synthesized six overlapping peptide amides corresponding to residues 106–167 of leptin, and examined their effects on body weight and food intake in female C57BL/6J ob/ob mice. When compared with vehicle-injected control mice, weight gain by mice receiving 28 daily 1-mg ip injections of LEP-(106–120), LEP-(116–130), or LEP-(126–140) was significantly (P < 0.01) reduced with no apparent toxicity. Weight gain by mice receiving LEP-(136–150), LEP-(146–160), or LEP-(156–167) was not significantly different from that of vehicle-injected control mice. The effects of LEP-(106–120), LEP-(116–130), or LEP-(126–140) were most pronounced during the first week of ...

Epitope mapping of secreted mouse leptin utilizing peripherally administered synthetic peptides

We have recently reported that intraperitoneal (i.p.) administration of synthetic peptide amides corresponding to amino acids 106-140 of mouse leptin significantly reduced food intake and body weight gain in female C57BL/6J ob/ob mice. These results suggested that leptin activity was localized in domains toward its C-terminus between residues 106-140. In the present study, 14 overlapping peptides encompassing the complete sequence of secreted mouse leptin were synthesized, and their effects on body weight and food intake in female C57BL/6 J ob/ob mice were assessed. When given as seven daily 1-mg i.p. injections, only peptides corresponding to amino acids 106-120, 116-130 and 126-140 caused significant reductions in body weight and food intake. These results confirmed our earlier study and suggest that in contrast to the domain encompassed by amino acids 106-140, the N-terminal of mouse leptin between amino acids 21-105 may not contain functional epitopes that can be utilized as lead compounds in the development of peripherally administered bioactive peptide analogs or nonpeptide mimetics of leptin, which may have potential usefulness in treatment of the energy imbalance associated with obesity.

Leptin and the treatment of obesity: its current status

Leptin, the protein product of the ob gene, is primarily an adipocyte-secreted hormone, whose functional significance is rapidly expanding. Although early research efforts were focused on defining leptins role in reversing obesity in rodents, there is now substantial evidence indicating that its influence extends to several hypothalamic-pituitary-endocrine axes, including gonadal, adrenal, thyroid, growth hormone, and pancreatic islets. A role for leptin in hematopoiesis, angiogenesis, immune function, osteogenesis, and wound healing has also been documented. The results of recent clinical trials with recombinant human leptin indicated that its effectiveness in restoring energy balance and correcting obesity-related endocrinopathies in genetically obese rodent models extended only partially to the management of human obesity. New efforts in drug development have focused on leptin-related synthetic peptide agonists as potential anti-obesity pharmacophores.

[d-LEU-4]-OB3, a synthetic leptin agonist, improves hyperglycemic control in C57BL/6J ob/ob mice

We have recently shown that the activity of a synthetic peptide corresponding to amino acid residues 116-130 of secreted mouse leptin is contained in a restricted sequence at the amino terminus of the peptide, between residues 116-122 (Ser-Cys-Ser-Leu-Pro-Gln-Thr, OB3). Substitution of the Leu residue at position 4 of OB3 with its D-isomer ([D-Leu-4]-OB3) enhanced the ability of OB3 (1 mg/day, ip, 7 days) to reduce body weight gain, food and water intake, and serum glucose in female C57BL/6J ob/ob mice. In the present study, we have utilized a pair-feeding approach to demonstrate that the antihyperglycemic action of [D-Leu-4]-OB3 is not solely due to its effects on caloric intake. One group of female C57BL/6J ob/ob mice (n=6) was fed ad libitum, and two additional groups (n=6 per group) were allowed 3.0 g food/mouse daily, an amount previously determined to satisfy [D-Leu-4]-OB3-treated mice. At the end of the 7-day test period, vehicle-injected mice fed ad libitum were approximately 10% heavier than their initial body weights, while pair-fed mice injected with vehicle and [D-Leu-4]-OB3-treated mice lost 5% of their initial body weights. After 1 day of treatment, blood glucose was reduced by 20% in pair-fed vehicle-injected mice, and by 40% in mice given [D-Leu-4]-OB3. Food restriction reduced blood glucose throughout the 7-day study, but not to levels seen in wild-type nonobese C57BL/6J mice of the same sex and age. After 2 days of treatment with [D-Leu-4]-OB3, however, blood glucose was reduced to levels comparable to those seen in wild-type nonobese mice. [D-Leu-4]-OB3 also lowered serum insulin levels by 53% when compared to mice fed ad libitum. Neither pair-feeding nor [D-Leu-4]-OB3 treatment had any apparent effect on thermogenesis. These results suggest that [D-Leu-4]-OB3 exerts its effects on serum glucose not only by suppressing caloric intake, but also through a separate effect on glucose metabolism which may involve increased tissue sensitivity to insulin.

Oral delivery of octreotide acetate in Intravail® improves uptake, half-life, and bioavailability over subcutaneous administration in male Swiss Webster mice

The most effective option for the medical treatment of patients with acromegaly is the use of somatostatin analogs. Octreotide acetate is a synthetic analog of somatostatin, with similar effects but a prolonged duration of action. Octreotide acetate is routinely given by subcutaneous (s.c.) or intramuscular injection. In the present study, we examined the feasibility of oral delivery of octreotide acetate reconstituted with increasing concentrations (0.5%, 1.5% and 3.0%) of Intravail®, a patented alkylsaccharide transmucosal absorption enhancing agent. The pharmacokinetics of orally delivered (by gavage) octreotide acetate in Intravail® were compared to those of octreotide acetate administered subcutaneously in sodium acetate buffer to male Swiss Webster mice. Oral delivery of octreotide acetate in 0.5% Intravail® significantly enhanced total uptake (1254.08ng/ml/min vs. 311.63ng/ml/min, respectively), serum half-life (52.1min vs. 1.3min, respectively), and relative bioavailability (4.0 vs. 1.0, respectively) when compared to delivery by s.c. injection. Higher concentrations of Intravail ®did not further enhance uptake, serum half-life, or bioavailability. The results of this study indicate that oral delivery of octreotide acetate in Intravail®is feasible, and is an effective method of administration which significantly improves uptake, bioavailability and half-life when compared to s.c. injection. Thus, oral delivery of octreotide acetate in Intravail® may have significant potential as a novel, non-invasive approach to the treatment of acromegaly and octreotide-mediated symptoms of carcinoid and VIP-secreting tumors.

Intranasal administration of mouse [D-Leu-4]OB3, a synthetic peptide amide with leptin-like activity, enhances total uptake and bioavailability in Swiss Webster mice when compared to intraperitoneal, subcutaneous, and intramuscular delivery systems.

Using a synthetic peptide strategy, we localized an active domain in mouse leptin to a sequence between amino acids 106 and 140. Intraperitoneal (ip) administration of a number of synthetic peptide amides encompassed by this domain reduced body weight gain, food and water intake, blood glucose levels, and increased insulin sensitivity in genetically obese mice. In the present study, we examined the pharmacokinetics of mouse [D-Leu-4]OB3, our most potent peptide, in male Swiss Webster mice following ip, subcutaneous (sc), and intramuscular (im) injection, and after intranasal administration with Intravail, a new class of patented transmucosal delivery enhancement agents. Total uptake (1,072,270, 1,182,498; 1,481,060; ng/ml/min), serum half-life (48.8; 34.0; 30.0 min) and relative bioavailability (1.0, 1.1; 1.4;) of mouse [D-Leu-4]OB3 were similar when the peptide was given by ip, sc, or im injection, respectively. Total uptake and relative bioavailability were enhanced following intranasal delivery (4,336,963 ng/ml/min and 4.0, respectively), and serum half-life was 41.1 min. These results indicate that intranasal delivery of mouse [D-Leu-4]OB3 with Intravail is a more effective method of peptide administration than injection methods, and suggest that it may have potential as a novel, non-invasive approach to the treatment of obesity and its associated metabolic dysfunctions in humans.

Oral delivery of mouse [D-Leu-4]-OB3, a synthetic peptide amide with leptin-like activity, in male Swiss Webster mice: a study comparing the pharmacokinetics of oral delivery to intraperitoneal, subcutaneous, intramuscular, and intranasal administration.

We have recently shown that intranasal administration of mouse [D-Leu-4]-OB3 reconstituted in Intravail to male Swiss Webster mice resulted in significantly higher bioavailability than commonly used injection methods of delivery. The absorption profile associated with intranasal delivery of mouse [D-Leu-4]-OB3 showed an early peak representing rapid uptake across the nasal mucosa, and a later peak suggesting a gastrointestinal site of absorption. In the present study, we show that gastrointestinal absorption of mouse [D-Leu-4]-OB3 does occur, and that reformulation of mouse [D-Leu-4-OB3 with Intravail significantly enhances its uptake. The pharmacokinetics of orally delivered (by gavage) mouse [D-Leu-4]-OB3 in the absence or presence of Intravail were examined, and compared to previously reported pharmacokinetic parameters of mouse [D-Leu-4]-OB3 following intraperitoneal (ip), subcutaneous (sc), intramuscular (im), and intranasal administration. When compared to oral delivery in PBS, Intravai significantly enhanced the total uptake (552,710 ng/ml/min vs.137,585 ng/ml/min) and relative bioavailability (4.0 vs. 1.0) of mouse [D-Leu-4-OB3. The relative oral bioavailabilities of mouse [D-Leu-4]-OB3 when compared to ip, sc, im, and intranasal delivery were 52.2%, 47.3%, 37.8% and 12.9%, respectively. The results of this study indicate that oral delivery of mouse [D-Leu-4]-OB3 in Intravail is an effective method of administration achieving relatively high serum levels of the bioactive peptide when compared to commonly used methods of injection. In addition to intranasal administration, oral delivery of mouse [D-Leu-4]-OB3 in Intravail may have potential as a novel, non-invasive approach to the treatment of obesity and its associated metabolic dysfunctions in humans.

Oral delivery of mouse [d‐Leu‐4]‐OB3, a synthetic peptide amide with leptin‐like activity, in male C57BL/6J wild‐type and ob/ob mice: effects on energy balance, glycaemic control and serum osteocalcin levels

Background: We have recently shown that intranasal administration of mouse [d‐Leu‐4]‐OB3 reconstituted in Intravail® to male Swiss Webster mice resulted in significantly higher bioavailability than commonly used injections methods of delivery. The absorption profile associated with intranasal delivery of mouse [d‐Leu‐4]‐OB3 showed an early peak representing absorption across the nasal mucosa, and a later peak suggesting a gastrointestinal site of uptake.

A synthetic peptide corresponding to the third cytoplasmic loop (residues 533 to 555) of the testicular follicle-stimulating hormone receptor affects signal transduction in rat testis membranes and in intact cultured rat Sertoli cells

Involvement of the third cytoplasmic (3i) loop (residues 533 to 555) of the rat testicular FSH receptor in the mechanism of FSH signal transduction was examined using light membranes prepared from immature rat testes, monolayer cultures of rat Sertoli cells, and a synthetic peptide strategy. This region of the FSH receptor is structurally related to G protein-activator regions identified in other G protein-coupled receptors. FSHR-(533-555) peptide amide stimulated guanine nucleotide exchange in rat testis light membranes, presumably via its interaction with membrane-associated G protein. The peptide failed to inhibit FSH binding to testis membrane receptors, indicating that the nucleotide exchange effect was not a result of peptide interaction with receptor. When incubated with cultured Sertoli cells from immature rat testes, FSHR-(533-555) peptide amide consistently and significantly inhibited FSH stimulation of cAMP and estradiol biosynthesis, but failed to inhibit forskolin stimulation of each. The peptide effect, therefore, was not due to a direct interaction with adenylyl cyclase. Since FSHR-(533-555) peptide amide did not inhibit FSH binding to membrane receptor, these results imply entry of the peptide into the Sertoli cell, possibly by vesicular internalization or diffusion. Indeed, the inhibitory effects of FSHR-(533-555) peptide amide on FSH-stimulated estradiol biosynthesis were prevented by pretreating Sertoli cells with phenylarsine oxide, an inhibitor of FSH receptor internalization. FSHR-(533-555) was without effect on basal levels of cAMP and estradiol biosynthesis, indicating absence of toxicity at the concentrations tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-function relationships of the glycoprotein hormones and their receptors

The primary structures of the glycoprotein hormones follitropin (FSH), lutropin (LH), human choriogonadotropin (hCG) and thyrotropin (TSH) have been determined, hCG has been crystallized and initial diffraction data obtained. Studies with synthetic peptides have provided information on regions involved in receptor interaction and signal transduction. The receptors for the glycoprotein hormones have been prepared by gene cloning methods and their primary structures deduced. As Leo Reichert and colleagues discuss here, although cAMP is involved in glycoprotein hormone signal transduction, recent evidence also implicates other second messengers, especially Ca2+ and may include both the phosphatidylinositol pathway and activation of Ca2+ channels.