Appetite Suppressant Activity Research Articles

TrkB-expressing paraventricular hypothalamic neurons suppress appetite through multiple neurocircuits

The TrkB receptor is critical for the control of energy balance, as mutations in its gene (NTRK2) lead to hyperphagia and severe obesity. The main neural substrate mediating the appetite-suppressing activity of TrkB, however, remains unknown. Here, we demonstrate that selective Ntrk2 deletion within paraventricular hypothalamus (PVH) leads to severe hyperphagic obesity. Furthermore, chemogenetic activation or inhibition of TrkB-expressing PVH (PVHTrkB) neurons suppresses or increases food intake, respectively. PVHTrkB neurons project to multiple brain regions, including ventromedial hypothalamus (VMH) and lateral parabrachial nucleus (LPBN). We find that PVHTrkB neurons projecting to LPBN are distinct from those to VMH, yet Ntrk2 deletion in PVH neurons projecting to either VMH or LPBN results in hyperphagia and obesity. Additionally, TrkB activation with BDNF increases firing of these PVH neurons. Therefore, TrkB signaling is a key regulator of a previously uncharacterized neuronal population within the PVH that impinges upon multiple circuits to govern appetite.

Morus alba, a Medicinal Plant for Appetite Suppression and Weight Loss.

The prevalence of obesity is expanding rapidly worldwide, making the disease a global burden with limited treatment options. The current obesity drug development trends suggest the possibility of reducing weight and reverse metabolic disturbances of obesity by controlling appetite. In this study, we screened more than 8000 plants from our plant library for the cannabinoid (CB1) receptor antagonists and identified Morus alba as a lead medicinal plant. Kuwanon G and Albanin G were isolated and identified from root-barks of Morus alba with 92% and 96% CB1 receptor ligand binding inhibitory activity, respectively. The bioflavonoid standardized extract was tested in the acute food intake study in rats at oral doses of 250 and 500 mg/kg for its appetite suppression activity. Diet-induced obesity in the C57BL/6J mice was used to evaluate the long-term food intake reduction activity and effect on the weight loss administered orally at 250 and 500 mg/kg for 7 weeks. Statistically significant and dose-dependent reduction in food intake was observed in both acute and long-term studies for the extract. Food intake reductions of 58.6% and 44.8% at 250 mg/kg and 50.1% and 44.3% at 500 mg/kg were observed at 1 and 2 h postfood provision, respectively. A 20% reduction in daily calorie intake was observed in the long-term study. Obese mice treated with the high dose of Morus root-bark extract showed 10.4 g (22.5%) and 7.1 g (16.5%) loss in body weight compared with the vehicle-treated obese animals (at week 7) and baseline, respectively. Statistically significant reductions in biochemical markers and visceral fat deposit were also observed. These results demonstrated that Morus alba extracts enriched in Kuwanon G, and Albanin G could be used alone to control appetite, manage body weight, and improve metabolic syndromes.

AgRP Neurons Can Increase Food Intake during Conditions of Appetite Suppression and Inhibit Anorexigenic Parabrachial Neurons.

To maintain energy homeostasis, orexigenic (appetite-inducing) and anorexigenic (appetite suppressing) brain systems functionally interact to regulate food intake. Within the hypothalamus, neurons that express agouti-related protein (AgRP) sense orexigenic factors and orchestrate an increase in food-seeking behavior. In contrast, calcitonin gene-related peptide (CGRP)-expressing neurons in the parabrachial nucleus (PBN) suppress feeding. PBN CGRP neurons become active in response to anorexigenic hormones released following a meal, including amylin, secreted by the pancreas, and cholecystokinin (CCK), secreted by the small intestine. Additionally, exogenous compounds, such as lithium chloride (LiCl), a salt that creates gastric discomfort, and lipopolysaccharide (LPS), a bacterial cell wall component that induces inflammation, exert appetite-suppressing effects and activate PBN CGRP neurons. The effects of increasing the homeostatic drive to eat on feeding behavior during appetite suppressing conditions are unknown. Here, we show in mice that food deprivation or optogenetic activation of AgRP neurons induces feeding to overcome the appetite suppressing effects of amylin, CCK, and LiCl, but not LPS. AgRP neuron photostimulation can also increase feeding during chemogenetic-mediated stimulation of PBN CGRP neurons. AgRP neuron stimulation reduces Fos expression in PBN CGRP neurons across all conditions. Finally, stimulation of projections from AgRP neurons to the PBN increases feeding following administration of amylin, CCK, and LiCl, but not LPS. These results demonstrate that AgRP neurons are sufficient to increase feeding during noninflammatory-based appetite suppression and to decrease activity in anorexigenic PBN CGRP neurons, thereby increasing food intake during homeostatic need.SIGNIFICANCE STATEMENT The motivation to eat depends on the relative balance of activity in distinct brain regions that induce or suppress appetite. An abnormal amount of activity in neurons that induce appetite can cause obesity, whereas an abnormal amount of activity in neurons that suppress appetite can cause malnutrition and a severe reduction in body weight. The purpose of this study was to determine whether a population of neurons known to induce appetite ("AgRP neurons") could induce food intake to overcome appetite-suppression following administration of various appetite-suppressing compounds. We found that stimulating AgRP neurons could overcome various forms of appetite suppression and decrease neural activity in a separate population of appetite-suppressing neurons, providing new insights into how the brain regulates food intake.

Antifeedant and repellent activities of Melia azedarach L. fruit extracts on fall armyworm

The aim of this study was to evaluate the appetite suppression and insecticidal activity of Melia azedarach aerial component extracts on the larvae of Spodoptera frugiperda obtained from the maceration by solvent, ultrasound and supercritical CO 2 extraction. Extracts of aerial components of M. azedarach were obtained by three different extraction methods (maceration, ultrasound and supercritical extraction) using different solvents (ethanol, n-hexane, ethyl acetate and CO 2 ) for use in testing appetite suppression activity in S.  frugiperda (Lepidoptera: Noctuidae). Extracts were sprayed on third instar larvae, which were then placed on corn seedlings. Extracts prepared by ethanol maceration of the fruits and branches showed appetite suppression and repellent activity, extracts of fruits obtained with n-hexane solvent and supercritical CO 2 resulted in significantly reduced leaf damage in the corn, with a result equivalent to the positive control (commercial insecticide). Regarding larvae mortality, the supercritical CO 2 extract of fruits resulted in the highest S. frugiperda mortality among the tested extracts, equivalent to treatment with positive control. This extract appears to be the most promising for controlling this corn crop insect pest.

Appetite Suppression and Antiobesity Effect of a Botanical Composition Composed of Morus alba, Yerba mate, and Magnolia officinalis.

Background. Obesity and its comorbidities continue to challenge the world at an alarming rate. Although the long term solution lies on lifestyle changes in the form of dieting and exercising, drug, medical food, or dietary supplement interventions are required for those who are already obese. Here we describe a standardized blend composed of extracts from three medicinal plants: Morus alba, Yerba mate, and Magnolia officinalis for appetite suppression and metabolic disorders management. Method. Extracts were standardized to yield a composition designated as UP601. Appetite suppression activity was tested in acute feed intake rat model. Efficacy was evaluated in C57BL/6J mouse models treated with oral doses of 1.3 g/kg/day for 7 weeks. Orlistat at 40 mg/kg/day was used as a positive control. Body compositions of mice were assessed using a dual energy X-ray absorptiometry (DEXA). ELISA was done for insulin, leptin, and ghrelin level quantitation. Nonalcoholic steatohepatitis (NASH) scoring was conducted. Results. Marked acute hypophagia with 81.8, 75.3, 43.9, and 30.9% reductions in food intake at 2, 4, 6, and 24 hours were observed for UP601. Decreases in body weight gain (21.5% compared to the HFD at weeks 7 and 8.2% compared to baseline) and calorie intake (40.5% for the first week) were observed. 75.9% and 46.8% reductions in insulin and leptin, respectively, 4.2-fold increase in ghrelin level, and reductions of 18.6% in cholesterol and 59% in low-density lipoprotein were documented. A percentage body fat of 18.9%, 47.8%, 46.1%, and 30.4% was found for mice treated with normal control, HFD, Orlistat, and UP601, respectively. 59.3% less mesenteric fat pad and improved NASH scores were observed for UP601. Conclusion. UP601, a standardized botanical composition from Morus alba, Yerba mate, and Magnolia officinalis could be used as a natural alternative for appetite suppression, maintaining healthy body weight and metabolism management.

Appetite Suppressing Phyto Nutrients: Potential for Combating Obesity

An appetite suppressant can be an excellent tool in weight management if used safely and effectively. There are some medicinal plants that have been reported to be acting as appetite suppressors or appetite killers. Some of these plants are Caralluma Fimbriata, Hoodia Gordonii, Camellia sinensis (Green Tea), Capsicum annuum (Sweet/Bell Pepper), Plantago ovata (Psyllium/Plantain Fiber), Saffron Extract, XanoLean, Flaxseeds, Ginger, Umeboshi Plums, Prunus dulcis (Almonds), Ilex paraguariensis (Yerba Mate) and Garcinia Cambogia, Aloe vera, Bee Pollen and Evening Primrose Oil. These types of herbal plants/supplements can be especially useful for people suffering from obesity for their weight management. Obesity increases risk of diseases and health problems, such as heart disease, diabetes and high blood pressure. This paper therefore aims to explore and provide an insight about the medicinal plants with appetite suppressing activity.

Metabolic response of short term calorie restriction and supplementation with Hoodia gordonii

Hoodia gordonii is a supplement of natural origin which is known for its appetite suppressant activity. Owing to its anorectic activity, the aim of this study was to evaluate the effect of H. gordonii supplementation on metabolic changes and appetite regulatory peptides during calorie restriction. Male albino rats were divided into three groups (n=12 in each) — Control, Calorie Restricted (CR, 25% for 5days), Calorie Restricted and H. gordonii supplemented (CR+HG, organic solvent extract given orally for 5days at a dose of 100mg/kg bwt.). The regulatory peptides i.e. ghrelin, leptin, CCK, NPY, insulin, IGF-1, corticosterone, thyroid hormones, adiponectin, serotonin were determined. On comparison with CR rats, modulations were noticed in the appetite regulatory peptides and biochemical variables of the CR+HG rats. A significant decline in ghrelin and increase in CCK was observed. The CR group exhibited a significant decrease in leptin, IGF-1, plasma and whole brain serotonin with a significant increase in the ghrelin and thyroxin levels. These changes indicate altered metabolic responses and hunger suppression which seem to be caused by H. gordonii with CR along with the changes occurring due to CR itself. It is concluded that H. gordonii can modulate hunger during CR and may be used for better adherence to dietary restriction regime.

Cultivation practices and manufacturing processes to produce Hoodia gordonii extract for weight management products

Hoodia gordonii (Masson) Sweet ex Decne., is a succulent shrub, indigenous to the arid regions of southern Africa. Indigenous people have historically utilised certain species of Hoodia, including H. gordonii, as a source of food and water. Studies by the Council for Scientific and Industrial Research (CSIR, South Africa) identified that extracts of H. gordonii had appetite suppressant activity associated with specific steroid glycosides. A programme to develop weight management products based around this discovery was implemented in 1998. An agronomy programme was established which demonstrated that it was possible to cultivate this novel crop on a commercial scale (in excess of 70ha). In parallel, a food grade manufacturing process was developed consisting of four main steps: harvesting of H. gordonii plant stems, comminution, drying under controlled conditions and extraction using food grade solvents. Appropriate Quality Control (QC) procedures were developed. The extraction process is capable of delivering a consistent composition despite natural variations in the composition of the dried H. gordonii. Specifications were developed for the resulting extract. The intended use of the standardised H. gordonii extract was as a functional food ingredient for weight management products. Other development studies on characterisation, toxicology and pharmacology are reported separately.

Synthesis and resolution of the novel appetite suppressant 2-benzylmorpholine, a nonstimulant isomer of phenmetrazine

The synthesis of 2-benzylmorpholine from allylbenzene together with its resolution into its (+)- and (-)-enantiomers is reported. Oral dosing of the racemate to dogs caused appetite suppression with an ED50 of 3 and 5.5 mg kg-1 at 1 and 2 h, respectively, after access to a meat meal. No stimulant activity was observed in dogs given oral doses of 200 mg kg-1 but the appetite suppressant effect in dogs declined during 20 days of chronic oral dosing at 10 mg kg-1. Appetite suppression activity was shown to reside in the (+)-enantiomer.

Bioavailability, Pharmacokinetics, and Tissue Distribution of the Oxypregnane Steroidal Glycoside P57AS3 (P57) fromHoodia gordoniiin Mouse Model

P57AS3 (P57), an oxypregnane steroidal glycoside, is known to be responsible for the appetite suppressing activity of HOODIA GORDONII, a dietary supplement used for weight loss. In this study, bioavailability, pharmacokinetics, and tissue distribution of P57 were determined in CD1 female mice after administration of a single dose of enriched methanolic extract of HOODIA GORDONII (equivalent to a dose of 25 mg of P57/kg) by oral gavage or a single dose of purified P57 (25 mg/kg) intravenously. The level of P57 in plasma and tissues (brain, liver, kidney, and intestine) was determined by UPLC-MS. After oral administration of HOODIA extract, the peak plasma level of P57 was achieved in 0.6 h. Upon intravenous administration, the plasma clearance rate of P57 was 1.09 L/h/kg. P57 was rapidly distributed and eliminated from the tissues within 4 hours. The level of tissue distribution was highest in the kidney followed by liver and brain. Upon oral administration, P57 was not detected in the brain and a very low concentration was seen in the intestine, kidney, and liver. Tissue/plasma ratio was 0.33 for brain, 0.57 for liver, and 0.75 for kidney with IV route and 0.11 for intestine, 0.02 for liver, and 0.04 for kidney with oral route. The half-life of the elimination phase was similar with both routes. The oral bioavailability was 47.5 % and the half-life of the absorption phase was 0.13 h. In conclusion, P57 showed moderate bioavailability and was eliminated rapidly.

Characterization of ADME Properties of P57AS3 and its Aglycone from Hoodia gordonii

This study was carried out to determine the ADME properties of oxypregnane steroidal glycoside P57AS3 (P57) and its aglycone from Hoodia gordonii by a series of in vitro assays to predict its gastric, intestinal and metabolic stability, intestinal and blood brain barrier (BBB) transport, protein binding and interaction with major drug metabolising enzymes CYP 3A4 and 2D6. Stability was determined in simulated gastric and intestinal fluids (SGF pH1.2 and SIF pH 6.8). Intestinal and BBB transports were monitored in Caco-2 and MDR-MDCK models. Phase I and phase II metabolic stability was determined in human liver microsomes and S9 fractions. Protein binding was determined by equilibrium dialysis. Quantitative analysis of P57 and aglycone was performed by HPLC. Inhibition of CYP 3A4 and 2D6 was determined by using cDNA overexpressed enzymes and fluorescence substrates. P57 was highly unstable in SGF (45% degradation in 30 minutes) while the corresponding aglycone was stable (4% degradation in 120 minutes). P57 and aglycone both were stable in the SIF. The intestinal transport of P57 was mediated by P-gp and MRP transporters, whereas aglycone was transported by a simple passive diffusion. No transport was observed for P57 across BBB whereas the aglycone was transported efficiently. P57 was highly stable in liver microsomes and S9 fractions whereas aglycone was not metabolically stable with a t1/2 of less than 15 minutes in the S9 fraction. P57 and aglycone were bound to the plasma proteins to an extent of 99.5 and 95%, respectively. P57 weakly inhibited CYP3A4 (IC50 33 µM) whereas aglycone strongly inhibited CYP3A4 (IC50 3 µM). No inhibition was observed for 2D6. The aglycone displayed better ADME properties compared to the glycoside. Although glycoside is considered to be responsible for appetite suppressant activity, aglycone seems to be transported through intestinal mucosa and BBB. Inhibition of CYP3A4 by the aglycone indicated the possibility of drug-drug interactions. Acknowledgements: We thank Mr. John Trott for excellent technical help. USDA, Agriculture Research Service Specific Cooperative Agreement No 58-6408-2-0009 and FDA, 2 U01 FD 002071-06 are acknowledged for partial support of this work.

Synthesis and appetite suppressant activity of 1-aryloxy-2-substituted aminomethyltetrahydronaphthalenes as conformationally rigid analogues of fluoxetine

Several 1-aryloxy-2-substituted aminomethyltetrahydronaphthalenes ( 7– 21) as conformationally rigid analogues of fluoxetine were synthesized and evaluated for their anorexigenic and antidepressant activities. For SAR studies the related acyclic analogues ( 22– 27) were also prepared. Out of the 21 synthesized compounds, 10 compounds ( 9, 10, 11, 15, 16, 18, 21, 22, 23 and 27) exhibited significant anorexigenic activity (at 75 μmol/kg). Interestingly, all the compounds ( 7– 20, 22– 26) were devoid of antidepressant effect, except for compounds 21 and 27 in which the antidepressant activity was retained. Compound 16 emerged as the most active compound of the series with better anorexigenic activity (97.92%) compared to fluoxetine (76.25%) and even with a clinically used drug sibutramine, thus providing a new structural lead for appetite suppressants.

Mechanism of simmondsin decomposition during sodium hydroxide treatment.

Jojoba seed meal shows appetite-suppressing activity due to the presence of simmondsin. This pharmacological activity disappears with treatment of the meal with sodium hydroxide. To elucidate this mechanism of inactivation, the reaction of simmondsin in 1 N NaOH at 20 degrees C was monitored as a function of time. The end products of the reaction as well as intermediates were isolated and identified. The half-life of simmondsin was approximately 60 min with d-glucose and 2-hydroxy-3-methoxyphenylacetonitrile as reaction end products. The reaction mechanism could be elucidated by the isolation of isosimmondsin and a simmondsin lactone derivative. Those compounds were isolated and purified by a combination of column chromatography and HPLC and identified mainly by HRMS and NMR spectroscopy.

2. The Effect of Feeding Excess Methionine on Live Performance, Carcass Traits, and Ascitic Mortality

Appetite suppressant activity of dietary excess Met was investigated in a 7-wk trial for the potential to restrict feed intake and the early growth of broilers as a means of reducing the incidence of ascitic mortality. A basal starter diet meeting the minimum NRC (1994) requirements for broilers was compared with a diet providing Met above the requirement (1.86% of the diet). Excess dietary Met was fed to the broilers from 4 to 11 d. Grower and finisher diets were provided from 22 to 35 d and from 36 to 49 d. Birds in two rooms were exposed to cool temperatures to induce ascites. The temperature program for the cool rooms was as follows: 12 h at 24 C daily (0800 to 2000) and 12 h at 15 C nightly (2000 to 0800) from 14 to 49 d, whereas the other two rooms were maintained at 24 C (control rooms). Body weight gain was depressed by 20.4% when excess Met was fed from 4 to 11 d. Feed conversion was also negatively affected by the excess dietary Met. A brief recovery was achieved from 11 to 14 d upon refeeding the well-balanced basal starter diet. Full compensatory growth was achieved by 49 d when body weight was equal to that of birds fed the basal diet. Excess dietary Met did not affect the carcass traits with the exception of abdominal fat pad, which was reduced by the excess dietary Met in the control rooms, whereas the opposite was true in the cool rooms. Ascitic mortality was not altered by dietary Met, however, the total mortality was significantly reduced by excess Met in the control rooms.

1. Appetite Suppressant Activity of Supplemental Dietary Amino Acids and Subsequent Compensatory Growth of Broilers

This study was conducted to take advantage of the appetite-suppressant effect of excessive dietary amino acids in reducing feed intake and, in turn, restricting the early rapid growth of broilers to minimize metabolic disorders. Dietary amino acids were supplemented to a basal diet to yield a total of 1.57, 2.57, and 3.57% His; 2.7, 4.3, and 5.9% Lys; 1.36, 2.16, and 2.96% Met; 2.8, 3.8, and 4.8% Thr; and 1.27, 2.27, and 3.27% Trp and were fed to 408 chicks from 4 to 11 d of age. Fifteen dietary treatments of His, Lys, Met, Thr, and Trp were compared to the basal diet. Feed consumption was measured daily. Body weight measurements were taken at 0, 4, 7, 11, 14, and 21 d. At 21 d, pectoralis major and minor muscles, liver, and abdominal fat pad were weighed.High levels of Met and His caused the greatest depression in appetite from 4 to 11 d, and Thr, Trp, and Lys were found to be less potent. The exponential growth rate (EGR) of birds from 4 to 11 d of age was significantly reduced by the intermediate and high levels of the amino acid supplementation. From 11 to 14 d, EGR was greatest with high levels of Met or Trp, indicating more potential compensatory growth realized with these treatments. The high level of His decreased the percentage of pectoralis minor muscle yield, whereas the high level of Lys and Met increased the percentage of liver compared to those fed the basal diet. These results indicate that it is possible to use excessive individual amino acids in diets to suppress the appetite and early rapid growth to alleviate or minimize metabolic disorders.

Leptin increases serotonin turnover by inhibition of brain nitric oxide synthesis

Leptin administration inhibits diencephalic nitric oxide synthase (NOS) activity and increases brain serotonin (5-HT) metabolism in mice. We evaluated food intake, body-weight gain, diencephalic NOS activity, and diencephalic content of tryptophan (TRP), 5-HT, hydroxyindoleacetic acid (5-HIAA), and 5-HIAA/5-HT ratio after intracerebroventricular (ICV) or intraperitoneal (IP) leptin injection in mice. Five consecutive days of ICV or IP leptin injections induced a significant reduction in neuronal NOS (nNOS) activity, and caused a dose-dependent increase of 5-HT, 5-HIAA, and the 5-HIAA/5-HT ratio. Diencephalic 5-HT metabolism showed a significant increase in 5-HT, 5-HIAA, and the 5-HIAA/5-HT ratio 3 hours after a single leptin injection. This effect was maintained for 3 hours and had disappeared by 12 hours after injection. After a single IP leptin injection, the peak for 5-HT, 5-HIAA, and the 5-HIAA/5-HT ratio was achieved at 6 hours. Single injections of ICV or IP leptin significantly increased diencephalic 5-HT content. Leptin-induced 5-HT increase was antagonized by the coadministration of L-arginine only when the latter was ICV injected, whereas D-arginine did not influence leptin effects on brain 5-HT content. Finally, in nNOS-knockout mice, the appetite-suppressant activity of leptin was strongly reduced, and the leptin-induced increase in brain 5-HT metabolism was completely abolished. Our results indicate that the L-arginine/NO pathway is involved in mediating leptin effects on feeding behavior, and demonstrate that nNOS activity is required for the effects of leptin on brain 5-HT turnover.

Tissue-specific processing of cocaine- and amphetamine-regulated transcript peptides in the rat.

Cocaine- and amphetamine-regulated transcript (CART) is a recently discovered hypothalamic peptide regulated by leptin and with a potent appetite-suppressing activity. In the rat, the CART gene encodes a peptide of 116 amino acid residues (or a splice variant 13 residues longer). The predicted signal sequence is 27 amino acid residues, resulting in a prohormone of 89 residues. The CART prohormone contains several potential posttranslational processing sites in the form of mono- and dibasic sequences. In the present study we have purified CART peptides from extracts of adrenal gland, hypothalamus, nucleus accumbens, and pituitary gland (anterior and neurointermediate lobe) of the rat and determined the peptide structures by using microsequencing and mass spectrometry. In none of the tissues examined the long splice variant was found. From the adrenal gland, the CART(1-89) and CART(10-89) peptides were isolated, in contrast to the hypothalamus and nucleus accumbens, from which the shorter form peptides CART(42-89) and CART(49-89) were purified. From the anterior lobe of the pituitary gland, CART(42-89) was isolated, in contrast to the neurointermediate lobe, which contains only CART(49-89). This tissue-specific processing indicates that CART peptides may have different biological functions in the periphery and in the central nervous system.

CART, a new anorectic peptide

Cocaine and amphetamine regulated transcript peptide (CART), is a recently discovered hypothalamic peptide with a potent appetite suppressing activity. In the rat the CART gene encodes a peptide of either 129 or 116 amino acid residues whereas only the short form exists in humans. The predicted signal sequence is 27 amino acid residues resulting in a prohormone of 102 or 89 residues. The C-terminal end of CART, consisting of 48 amino acid residues and 3 disulphide bonds, is thought to constitute a biologically active part of the molecule. In the central nervous system CART is highly expressed in many hypothalamic nuclei, some of which are involved in regulating feeding behaviour. The CART mRNA is regulated by leptin, and the expressed CART is a potent inhibitor of feeding that even overrides the feeding response induced by neuropeptide Y. The putative CART receptor is therefore a potential therapeutic target for an anti-obesity drug.

Purification and characterisation of a new hypothalamic satiety peptide, cocaine and amphetamine regulated transcript (CART), produced in yeast

Cocaine and amphetamine regulated transcript (CART) is a newly discovered hypothalamic peptide with a potent appetite suppressing activity following intracerebroventricular administration. When the mature rat CART sequence encoding CART(1–102) was inserted in the yeast expression plasmid three CART peptides could be purified from the fermentation broth reflecting processing at dibasic sequences. None of these corresponded to the naturally occurring CART(55–102). In order to obtain CART(55–102) the precursor Glu-Glu-Ile-Asp-CART(55–102) has been produced and CART(55–102) was generated by digestion of the precursor with dipeptidylaminopeptidase-1. All four generated CART peptides have been characterised by N-terminal amino acid sequencing and mass spectrometry. The CART peptides contain six cysteine residues and using the yeast expressed CART(62–102) the disulphide bond configuration was found to be I–III, II–V and IV–VI. When the four CART peptides were intracerebroventricularly injected in fasted mice (0.1 to 2.0 μg) they all produced a dose dependent inhibition of food intake.

Synthesis and biological activity of CCK heptapeptide analogs. Effects of conformational constraints and standard modifications on receptor subtype selectivity, functional activity in vitro, and appetite suppression in vivo

A series of modifications of the CCK7 analogue (des-NH2)Tyr(SO3-)-Nle-Gly-Trp-Nle-Asp-Phe-NH2 was prepared and tested for binding to guinea pig CCK-A and CCK-B receptors and in CCK-A-mediated functional assays. Selected analogues also were tested for appetite suppressant activity in rats. Several conformationally restricted residues in the C-terminal tetrapeptide region, including delta Z-Phe33, (N-Me)Phe33, (N-Me)Asp32, (N-Me)Leu31, and 3PP31 (3PP = trans-3-n-propyl-L- proline) were found to be acceptable modifications at one or both receptor subtypes. The (N-Me)Asp32 and (N-Me)Leu31 modifications afforded potent and selective CCK-A and CCK-B ligands, respectively. SAR studies in the N-terminal acyldipeptide region examined structural requirements for the side chain at position 28, where Gly and Pro replacements were found to possess high affinity at both receptor subtypes. Other conformationally restrictive modifications were less active. All of the analogues that showed high affinity (less than 10 nM) for the CCK-A receptor also were full agonists in amylase release and most were full or nearly full agonists in the phosphoinositide (PI) turnover assay, the most notable exception being the delta Z-Phe33 analogue, which showed 69% of the maximal response in the PI assay. Potent activity in suppression of food intake in rats was found for selected analogues.