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Metabolic Studies of a Synthetic Lipolytic Domain (AOD9604) of Human Growth Hormone
Abstract
A synthetic analogue (AOD9604) of the lipolytic domain of human growth hormone (hGH) has been studied for its metabolic actions in obese Zucker rats. Daily treatment with an oral dose of AOD9604 of 500 microg/kg body weight for 19 days reduced over 50% (15.8 +/- 0.6 vs. 35.6 +/- 0.8 g) body weight gain of the animals in comparison with the control. The adipose tissues of the AOD9604--treated animals were found to have an increase in lipolytic activity. In contrast to chronic treatment with intact hGH, chronic treatment with AOD9604 showed no adverse effect on insulin sensitivity of the animals, as demonstrated with euglycemic clamp techniques. The results in the present study suggest that the analogue of the hGH lipolytic domain may have the potential to be developed into an orally usable and safe therapeutic agent for obesity.
... Insulin resistance increases fatty acid invasion in the liver in favor of lipogenesis and prevention of fats lipolysis [16]. However, growth hormone inhibits adipocyte differentiation, reduces triglycerides, and increases lipolysis through the G protein signaling pathway [17]. The 15 GH terminal amino acids stimulate lipolysis, which was first documented by pharmacological researchers at Monash University in Australia. ...
... Importantly, the fragment does not stimulate insulin resistance, so its lipolytic response is higher than the growth hormone. It is stated that it has no growth and synthesis effects like growth hormone [17]. ...
Aims: Fatty liver and its treatment are among the concerns of today’s society. So this study aimed to investigate the effect of endurance training, Somatropin injection, and its lipolytic fragment (AOD9604) on cytokeratin-18 (CK18) levels and liver enzymes of mice with fatty liver.
Methods & Materials: In this experimental study, 28 male mice were randomly divided into four groups (7 mice in every group): control (C), exercise (E), exercise + fragment (EA), and exercise + growth hormone (EGH). A medium-intensity endurance training program was performed for 8 weeks, 5 sessions per week with an intensity of 50% VO2max. The somatropin and fragment injection protocols were 1 mg and 250 mc/kg of body weight, respectively. The mice were evaluated 48 hours after the last training session. The obtained data were analyzed using 1-way ANOVA and post hoc Tukey tests at the significant level of P
... Interestingly, the carboxyl terminus (amino acids 177 -191) appears to be a lipid mobilizing domain with inhibitory action on the acetyl-CoA carboxylase activity in hepatocytes and adipocytes [31]. Several C-terminal fragments were synthesized and tested for their effects in a number of animal models of obesity. ...
... AOD9604 showed metabolic effects similar to those of intact hGH, with respect to energy balance and fat oxidation in chronically treated animals [36]. AOD9604 elicited the fat reducing effect without inducing insulin resistance [31]. AOD9604 did not stimulate the production of IGF-1 in any of the systems studied. ...
Background: AOD9604 is the C-terminal fragment (TyrhGH177-
191) of human growth hormone (hGH). Early stage studies
with AOD9604 have shown positive activities on fat metabolism.
As a result the use of AOD9604 to improve metabolic profiles
may be plausible. For these uses, the safety of AOD9604 needs to
be clearly demonstrated. Here we report the findings from studies
involving the genotoxicological, toxicological and pharmacokinetic
testing of AOD9604.
Methods: In an Ames test, the mutagenic potential of AOD9604
was assessed. A chromosomal aberration assay in CHO cells was
used to test the mutagenic activity of AOD9604 by its ability to
cause structural damage to chromosomes. A bone micronucleus assay
incorporated in a 4-week rat intravenous (IV) toxicity study was
used to test potential micronucleus formation. In chronic toxicology
studies in rats (6 months) and cynomolgus monkeys (9 months), the
potential toxicity of AOD9604 was assessed after daily oral gavage.
Pharmacokinetic properties of AOD9604 were examined in a study
with pigs after oral and IV administration, and rat whole-body radiography
after IV and oral 14C-AOD9604 application.
Results: AOD9604 was found to be generally safe after chronic
oral application in rats and cynomolgus monkeys. There was no evidence
of any genotoxic activities of AOD9604, as examined in an
Ames test, a chromosomal aberration assay, or a bone micronucleus
assay. Rat whole-body radiography revealed similar organ distribution
after IV or oral application. Orally administered AOD9604
in pigs was well absorbed and results revealed rapid degradation
kinetics.
Conclusion: Multiple non-clinical studies revealed no evidence of
genotoxicological or toxicological concerns regarding the safety of
AOD9604.
... The adipose tissues of the AOD9604treated animals were found to present an increase in lipolytic activity. In contrast to chronic treatment with intact hGH, the chronic treatment with AOD9604 showed no adverse effect on insulin sensitivity, as demonstrated with euglycemic clamp techniques [94]. Unfortunately, AOD9604 recently failed phase IIb trials for obesity. ...
... This analogue of the hGH lipolytic domain may have the potential to be developed into an orally usable and safe therapeutic agent for obesity. Up to now, no human studies have been yet performed [94]. ...
The prevalence of overweight and obesity is increasing in children and adolescents worldwide raising the question on the approach to this condition because of the potential morbidity, mortality, and economic tolls. Dietetic and behavioral treatments alone have only limited success; consequently, discussion on strategies for treating childhood and adolescent obesity has been promoted. Considering that our knowledge on the physiological systems regulating food intake and body weight is considerably increased, many studies have underlined the scientific and clinical relevance of potential treatments based on management of peripheral or central neuropeptides signals by drugs. In this paper, we analyze the data on the currently approved obesity pharmacological treatment suggesting the new potential drugs.
... 18 A C-terminal hGH fragment 176-191 with a tyrosine to phenylalanine substitution at the last position has been reported to enhance lipid breakdown and fat utilization in mice. [19][20][21] And hence, in the present study we examined if hGH fragment 176-191 peptide can facilitate the anticancer efficacy of doxorubicin-loaded nanoparticles. ...
Introduction: Numerous drugs with potent toxicity against cancer cells are available for treating malignancies, but therapeutic efficacies
are limited due to their inefficient tumor targeting and deleterious effects on non-cancerous tissue. Therefore, two improvements are
mandatory for improved chemotherapy 1) novel delivery techniques that can target cancer cells to deliver anticancer drugs and 2) methods to
specifically enhance drug efficacy within tumors. The loading of inert drug carriers with anticancer agents and peptides which are able to
bind (target) tumor-related proteins to enhance tumor drug accumulation and local cytotoxicity is a most promising approach.
Objective: To evaluate the anticancer efficacy of Chitosan nanoparticles loaded with human growth hormone hGH fragment 176–191
peptide plus the clinical chemotherapeutic doxorubicin in comparison with Chitosan loaded with doxorubicin alone.
Methods: Two sets of in silico experiments were performed using molecular docking simulations to determine the influence of hGH
fragment 176–191 peptide on the anticancer efficacy of doxorubicin 1) the binding affinities of hGH fragment 176–191 peptide to the breast
cancer receptors, 2) the effects of hGH fragment 176–191 peptide binding on doxorubicin binding to these same receptors. Further, the
influence of hGH fragment 176–191 peptide on the anticancer efficacy of doxorubicin was validated using viability assay in Human MCF-7
breast cancer cells.
Results: In silico analysis suggested that addition of the hGH fragment to doxorubicin-loaded Chitosan nanoparticles can enhance
doxorubicin binding to multiple breast cancer protein targets, while photon correlation spectroscopy revealed that the synthesized dualloaded
Chitosan nanoparticles possess clinically favorable particle size, polydispersity index, as well as zeta potential.
Conclusion: These dual-loaded Chitosan nanoparticles demonstrated greater anti-proliferative activity against a breast cancer cell line
(MCF-7) than doxorubicin-loaded Chitosan. This dual-loading strategy may enhance the anticancer potency of doxorubicin and reduce
the clinical side effects associated with non-target tissue exposure.
Keywords: anticancer potency, nanoparticles, cytotoxicity, docking analysis
... The exact mechanism underlying the action of GH in OA is unknown. Previous studies have shown that GH can act directly on the growth plate by stimulating local production of IGF-1 and by increasing cartilage metabolism [9,16] and chondrocyte proliferation [17]. Although AOD9604 is not a high-affinity agonist of the GH receptor and does not stimulate the proliferation of cells transfected with the GH receptor, it retains some functions of GH [11]. ...
To investigate the effects of AOD9604 intra-articular injections with or without hyaluronic acid (HA) in a collagenase-induced knee osteoarthritis (OA) rabbit model.
Mature New Zealand white rabbits (n=32) were randomly administered 2 mg collagenase type II twice in each knee joint. Weekly injections of 0.6 mL saline (Group 1), 6 mg HA (Group 2), 0.25 mg AOD9604 (Group 3), and 0.25 mg AOD9604 with 6 mg HA (Group 4) were administered for 4-7 weeks after the first intra-articular collagenase injection. The degree of cartilage degeneration was assessed using morphological and histopathological findings, and the degree of lameness was observed at 8 weeks after the first collagenase injection.
Mean gross morphological and histopathological scores were significantly higher in Group 1 than in Groups 2, 3, and 4, and the scores were significantly lower in Group 4 than in Groups 2 and 3. The lameness period in Group 4 was significantly shorter than those in Groups 1, 2, and 3. The lameness period in Group 1 was significantly longer than those in Groups 2 and 3.
Intra-articular AOD9604 injections using ultrasound guidance enhanced cartilage regeneration, and combined AOD9604 and HA injections were more effective than HA or AOD9604 injections alone in the collagenase-induced knee OA rabbit model.
© 2015 by the Association of Clinical Scientists, Inc.
... The present evidence suggests that treatment with AOD9401 did not affect insulin sensitivity or glucose tolerance of the animals under the experimental conditions as described. Recent glucose clamp experiments with an analogue compound (AOD9604) produced the same result (Ng et al. 2000). The plasma insulin levels of the experimental animals had not been measured as no significant effect of the AOD compound on the endogenous insulin levels had previously been detected in our obese mice studies. ...
A lipolytic domain (AOD9401) of human growth hormone (hGH) which resides in the carboxyl terminus of the molecule and contains the amino acid residues 177-191, has been synthesized using solid-phase peptide synthesis techniques. AOD9401 stimulated hormone-sensitive lipase and inhibited acetyl coenzyme A carboxylase (acetyl CoA carboxylase) in isolated rat adipose tissues, in a similar manner to the actions of the intact hGH molecule. The synthetic lipolytic domain mimicked the effect of the intact growth hormone on diacylglycerol release in adipocytes. Chronic treatment of obese Zucker rats with AOD9401 for 20 days reduced the body weight gain of the animals, and the average cell size of the adipocytes of the treated animals decreased from 110 to 80 microm in diameter. Unlike hGH, synthetic AOD9401 did not induce insulin resistance or glucose intolerance in the laboratory animals after chronic treatment. The results suggest that AOD9401 has the potential to be developed into a therapeutic agent for the control of obesity.
... [4] In rodent models of obesity, AOD9604 reduced body weight gain, increased one indicator of lipolysis, and increased fat oxidation when compared to treatment with saline. [5,6] Interestingly, the peptide did not compete with hGH for binding to the GH receptor. Instead, the long-term effects of the peptide were shown to require the presence of the β 3 -adrenergic receptor. ...
AOD9604 is a peptide consisting of the C-terminal fragment of human growth hormone from amino acids 177–191 with an additional tyrosine residue at the N-terminus of the peptide. It is reported to mimic the lipolytic properties of growth hormone without the diabetogenic side effects. Therefore, AOD9604 may be used as a performance enhancing drug and is banned by the World Anti-doping Agency (WADA). The peptide is available on several Internet websites and was recently identified in confiscated vials in the USA. To detect abuse of the peptide in athletes, a solid-phase extraction method was validated in urine with a limit of detection of 50 pg/mL. The method has good linearity, precision (<20%), specificity and recovery (62%). Six potential metabolites of the peptide were identified after incubation of AOD9604 in serum and urine. Quantification of the metabolites in serum identified a single metabolite, consisting of amino acids CRSVEGSCG, which is significantly more stable than the other metabolites or the parent compound. Screening for AOD9604 and the stable metabolite may potentially allow an increased window of detection. Copyright © 2014 John Wiley & Sons, Ltd.
... The amino-terminus of the hGH molecule is the functional domain for the insulin-like action of the hormone [281]. Some studies had identified the metabolic domain responsible specifically for the lipolytic/antilipogenic activity of the hGH molecule, referred as AOD9604 and it has been studied for its metabolic actions in obese rats. ...
The growing worldwide prevalence of obesity needs urgent attention because the potential morbidity, mortality, and economic tolls have to be avoided. Despite obesity is known as a healthcare issue on an epidemic scale, it remains largely an unsolved medical problem. The successful management of obesity is theoretically possible through lifestyle changes, with diet modifications and increasing physical activity. However, low results by traditional treatments have inevitably prompted interest in the development of effective therapies, including pharmacological interventions and gastrointestinal surgery. As our knowledge of the physiological systems regulating food intake and body weight has considerably increased over the past decade, many studies have underlined the scientific and clinical relevance of potential treatments based on peripheral hormones or central neuropeptides signals. Here we have summarized the complex pattern of the appetite regulation, divided into central and peripheral mechanisms. In the second part of this paper, we have reviewed the currently approved and putative obesity therapies. Up to now only two drugs, sibutramine and orlistat have been approved by the Food and Drug Administration for long term use, but several other medications are currently used to cure severe obesity and many other are developing. Thus, in the last part, we have analyzed recent literature and patents describing new and upcoming molecules. The new anti-obesity drugs under clinical development include agents affecting peripheral and central mechanisms. Further investigations are needed to approve these upcoming therapeutic agents for the treatment of obesity.
... AOD-9604 is an orally active analogue of peptide fragment of human growth hormone (hGH 177--191) that selectively activates lipolysis in adipose tissue. In one study, AOD-9604 reduced body weight in genetically obese zucker rats and ob/ob mice, without inducing the untoward effect on glycemic control normally observed with growth hormone [191]. In a 12-week randomized clinical trial, subjects receiving AOD-9604 (1 mg/d) lost an average of 2.6 kg, compared to 0.8 kg in the placebo group [192]. ...
The rising tide of obesity and its related disorders is one of the most pressing health concerns worldwide, yet existing medicines to combat the problem are disappointingly limited in number and effectiveness. Recent advances in mechanistic insights into the neuroendocrine regulation of body weight have revealed an expanding list of molecular targets for novel, rationally designed antiobesity pharmaceutical agents. Antiobesity drugs act via any of four mechanisms: 1) decreasing energy intake, 2) increasing energy expenditure or modulating lipid metabolism, 3) modulating fat stores or adipocyte differentiation, and 4) mimicking caloric restriction. Various novel drug candidates and targets directed against obesity are currently being explored. A few of them are also in the later phases of clinical trials. This review discusses the development of novel antiobesity drugs based on current understanding of energy homeostasis.
... 30 AOD9604, a modified fragment of the GH, has been evaluated in animal and early phase human studies. 31 It is administered orally and binds to the fat cell, stimulating lipolysis and inhibiting re-esterification without stimulating growth. However, a meta-analysis, including over 500 subjects, found these beneficial effects on adiposity and lipid profile to be quantitatively small compared with the supraphysiological dose used in many studies. ...
The increasing prevalence of obesity and associated morbidity present unmet medical needs for safe and effective new drug therapies. Our aim is to review the diverse targets and compounds that are in clinical development.
Literature searches were conducted using the PUBMED database for studies published in English from January 1985 to December 2011 using combinations of key words, including obesity, overweight, weight loss and treatment in addition to the clinical trials website. Bibliographies of selected references were also evaluated for relevant articles. Press/news releases were also utilized. The collection of information for this review was limited to the most recently available human and animal data.
Weight loss drugs in development include compounds that act centrally (neuropeptide Y, AgRP and MCH1 receptors) to limit food intake or reduce the absorption of fat from the gastrointestinal tract (lipase inhibitors) or increase energy expenditure or reduce adipose tissue formation. Among the existing therapy, new combinations (topiramate plus phentermine, bupropion plus naltrexone) offer greater efficacy with reduced adverse effects.
Despite recent setbacks in the pharmacotherapy of obesity (withdrawal of rimonabant and sibutramine), many compounds are in phase II/III trials. The future holds promise for a new drug that alone or in combination with an existing agent could target the initial pathophysiology and morbidities associated with obesity.
... A small region of the growth hormone molecule, denoted hGH 177-191, appears to retain some of the actions of growth hormone, but with no effect on growth or on insulin resistance. An orally active peptide variant of hGH 177-191, called AOD9604, was shown to stimulate metabolism of fat in animal trials [88,89] but, after a phase IIB clinical trial, the results did not support the commercial viability of the drug as a treatment for obesity. ...
Available anti-obesity pharmacotherapy options remain very limited and development of more effective drugs has become a priority. The potential strategies to achieve weight loss are to reduce energy intake by stimulating anorexigenic signals or by blocking orexigenic signals, and to increase energy expenditure. This review will focus on approved obesity medications, as well as potential new pharmacologic treatment options.
... The present evidence suggests that treatment with AOD9401 did not affect insulin sensitivity or glucose tolerance of the animals under the experimental conditions as described. Recent glucose clamp experiments with an analogue compound (AOD9604) produced the same result (Ng et al. 2000). The plasma insulin levels of the experimental animals had not been measured as no significant effect of the AOD compound on the endogenous insulin levels had previously been detected in our obese mice studies. ...
A lipolytic domain (AOD9401) of human growth hormone (hGH) which resides in the carboxyl terminus of the molecule and contains the amino acid residues 177-191, has been synthesized using solid-phase peptide synthesis techniques. AOD9401 stimulated hormone-sensitive lipase and inhibited acetyl coenzyme A carboxylase (acetyl CoA carboxylase) in isolated rat adipose tissues, in a similar manner to the actions of the intact hGH molecule. The synthetic lipolytic domain mimicked the effect of the intact growth hormone on diacylglycerol release in adipocytes. Chronic treatment of obese Zucker rats with AOD9401 for 20 days reduced the body weight gain of the animals, and the average cell size of the adipocytes of the treated animals decreased from 110 to 80 microm in diameter. Unlike hGH, synthetic AOD9401 did not induce insulin resistance or glucose intolerance in the laboratory animals after chronic treatment. The results suggest that AOD9401 has the potential to be developed into a therapeutic agent for the control of obesity.
... The parent molecule, AOD9401, induces lipolysis and antilipogenesis and fat oxidation in adipose tissue in vitro (8,9). In vivo, AOD9401 induces weight loss without affecting food intake as well as increasing lipolytic sensitivity and increasing fat oxidation with no adverse effects on insulin sensitivity (8,10). ...
Both human GH (hGH) and a lipolytic fragment (AOD9604) synthesized from its C-terminus are capable of inducing weight loss and increasing lipolytic sensitivity following long-term treatment in mice. One mechanism by which this may occur is through an interaction with the beta-adrenergic pathway, particularly with the beta(3)-adrenergic receptors (beta(3)-AR). Here we describe how hGH and AOD9604 can reduce body weight and body fat in obese mice following 14 d of chronic ip administration. These results correlate with increases in the level of expression of beta(3)-AR RNA, the major lipolytic receptor found in fat cells. Importantly, both hGH and AOD9604 are capable of increasing the repressed levels of beta(3)-AR RNA in obese mice to levels comparable with those in lean mice. The importance of beta(3)-AR was verified when long-term treatment with hGH and AOD9604 in beta(3)-AR knock-out mice failed to produce the change in body weight and increase in lipolysis that was observed in wild-type control mice. However, in an acute experiment, AOD9604 was capable of increasing energy expenditure and fat oxidation in the beta(3)-AR knock-out mice. In conclusion, this study demonstrates that the lipolytic actions of both hGH and AOD9604 are not mediated directly through the beta(3)-AR although both compounds increase beta(3)-AR expression, which may subsequently contribute to enhanced lipolytic sensitivity.
... The lipolytic effect was mediated by an increase in hormone-sensitive lipase (HSL) activity. The anti-lipogenic effect was caused, however, by significant reduction in acetyl-CoA carboxylase activity [25,26]. This effect was found to be dose dependent. ...
Understanding the relationship between growth hormone (GH) structure (its molecular fragments) and function via interaction with single or multiple receptors is of particular importance in clinical diagnostics and physiologic biochemistry. Direct and indirect actions of GH are numerous ranging from carbohydrate and lipid metabolism to growth effects at muscle and vessels. To this end, we have focused on the influence of physical exercise on GH synthesis and release into the circulation. Physical exercise is a physiological condition to which GH multifunctionality is inextricably linked and is thus important physiologically and pathologically. This review describes the potential human GH fragments with respect to protein hormone multifunctionality and the molecular regions of potential action. The intent of the review is to highlight human GH fragments and hypothesize their potential physiologic role. GH fragmentation is also reviewed in relation to the effects of physical exercise and hormone multifunctionality.
... AOD-9604 is a synthetic fragment of human GH, with a modified C-terminal sequence being developed by Metabolic Pharmaceuticals [82]. It stimulates lipolysis and inhibits triglyceride synthesis, without effects on cell proliferation (at least in BaF3 cells transfected with the GH receptor gene) or having adverse effects on insulin resistance in obese mice [83], in ob/ob and lean mice [84] and obese Zucker rats [85]. The results suggest that the analogue of the human GH lipolytic domain may have the potential to be developed into an orally usable and safe therapeutic agent for obesity. ...
Obesity is a major public health concern and environmental factors are involved in its development. The hypothalamus is a primary site for the integration of signals for the regulation of energy homeostasis. Dysregulation of these pathways can lead to weight loss or gain. Some drugs in development can have favourable effects on body weight, acting on some of these pathways and leading to responses resulting in weight loss. Strategies for the management of weight reduction include exercise, diet, behavioural therapy, drug therapy and surgery. Investigational antiobesity medications can modulate energy homeostasis by stimulating catabolic or inhibiting anabolic pathways. Investigational drugs stimulating catabolic pathways consist of leptin, agonists of melanocortin receptor-4, 5-HT and dopamine; bupropion, growth hormone fragments, cholecystokinin subtype 1 receptor agonist, peptide YY3-36, oxyntomodulin, ciliary neurotrophic factor analogue, beta3-adrenergic receptor agonists, adiponectin derivatives and glucagon-like peptide-1. On the other hand, investigational drugs inhibiting anabolic pathways consist of the ghrelin receptor, neuropeptide Y receptor and melanin-concentrating hormone-1 antagonists; somatostatin analogues, peroxisome proliferator-activated receptor-gamma and -beta/delta antagonists, gastric emptying retardation agents, pancreatic lipase inhibitors, topiramate and cannabinoid-1 receptor antagonists. These differing approaches are reviewed and commented on in this article.
Introduction: Fatty liver is on the rise in the world, so the aim of this study was to investigate The effect of eight weeks of endurance training, injection of growth hormone lipolytic fragment (AOD9604) on CK18 and liver enzymes of NAFLD on mice induced mice induced by high-fat diet. Methods: In This experimental study, 28 male mice were, divided into four groups(n=7): control
The chapters within this book have detailed various aspects of the neurobiology of weight control. These include the genetic factors which determined the function of the body's energy regulation and the central mechanisms responsible for maintaining the body's energy balance. Particular focus has been placed on central targets such as the melanocortin and endogenous opioid systems. These systems represent two factors which control food intake: Energy balance regulation and pleasure/reward. It is the metabolic demand for energy and the pleasure derived from eating palatable foods which determine when, what and how much we eat. Other chapters have dealt with peripheral generated signals such as ghrelin, leptin and insulin and their role in appetite and energy regulation. Such mechanisms provide episodic meal-by-meal signals of food consumption and the tonic signals of energy storage to the CNS. Organs such as the gut, the pancreas and adipose tissue act as both detectors and effectors in the organism energy regulation system. This diverse peripheral input allows the organism to constantly monitor its current energy status. In turn the CNS does not only adjust the expression of feeding behavior, as the last chapter shows the CNS also exerts control over the storage of energy. Given the complexity of these systems underpinning energy regulation (episodic and tonic, peripheral and central) it may appear surprising that the state of obesity exists. However, despite the collective action of these many systems it seems many individuals experience great difficulty controlling their own body weight
Currently available drug therapy of overweight and obesity is very limited, therefore development of more effective drugs has become an important goal. The two general strategies to achieve weight loss are reduction of energy intake by stimulation of anorexigenic signals or inhibition of orexigenic signals, and increase of energy expenditure. This review focuses on approved and recently removed anti-obesity drugs, as well as on new approaches for drug treatment of obesity.
Introduction:
As the first peptide HIV fusion inhibitor targeting gp41, enfuvirtide (T20) was approved by the US FDA in 2003 as a salvage therapy for HIV/AIDS patients who failed to respond to the then existing antiretroviral therapeutics. However, its clinical application is limited by its relatively low potency, low genetic barrier to drug resistance and short half-life. Therefore, it is essential to develop new peptide HIV fusion inhibitors with improved antiviral efficacy, drug-resistance profile and pharmaceutical properties.
Areas covered:
In this paper, we reviewed the patents, patent applications and related research articles for the development of new peptide fusion inhibitors targeting the HIV-1 gp41 published between 2009 and 2014.
Expert opinion:
To improve enfuvirtide's anti-HIV efficacy, drug-resistance profile, half-life and pharmaceutical properties, the best approaches include the addition of the pocket-binding domain (PBD) to the N-terminus of T20 and linking of the M-T hook to the N-terminus of PBD, as well as conjugation of cholesterol, serum albumin-binding motif or gp120-binding fragment with a PBD-containing C-terminal heptad repeat-peptide. Therefore, sifuvirtide from Tianjin FusoGen Pharmaceuticals, Inc., albuvirtide from Frontier Biotechnologies Co., Ltd., cholesterol-conjugated HIV fusion inhibitor from the Institute of Pathogen Biology, Chinese Academy of Medical Science, 2DLT, a bivalent HIV fusion inhibitor/inactivator, and an enfuvirtide/sifuvirtide combination regimen from the New York Blood Center may all have potential as next-generation HIV fusion inhibitors targeting gp41 for clinical use.
Growth hormone (GH) signalling is traditionally via a 'classical' GH-receptor (GHR) that is present in the plasma membrane of target cells. GHRs are present in the neural retina of early chick embryos (within the first trimester of incubation) but these are unlikely to be endocrine target sites of GH action, as pituitary GH is not secreted into the bloodstream until late in development (approximately day 17 of the 21 days incubation period) and because the blood–ocular barrier would block the entry of systemic GH into the retina. As GH is expressed in the early neural retina, retinal GHRs are thus likely to be autocrine or paracrine sites of GH action. The most abundant GH moieties in the neural retina are, however, unlikely to activate 'classical' GHRs in the neural retina, as they are submonomeric molecules (of 15 kDa and 16.5 kDa) that fail to bind to plasma membrane receptor (15 kDa GH) or lack the stoichiometric requirements likely required for GHR binding (16.5 kDa GH). Nevertheless, 15 kDa GH is biologically active in chicks and the immunoneutralisation of endogenous 16.5 kDa GH induces biological activity. It is therefore possible that GH in the neural retina of early chick embryos acts non-classically or via 'non-classical' GHRs. These possibilities are the focus of this brief review.
The number and diversity of potentially performance-enhancing substances is continuously growing, fueled by new pharmaceutical developments but also by the inventiveness and, at the same time, unscrupulousness of black-market (designer) drug producers and providers. In terms of sports drug testing, this situation necessitates reactive as well as proactive research and expansion of the analytical armamentarium to ensure timely, adequate, and comprehensive doping controls. This review summarizes literature published over the past 5 years on new drug entities, discontinued therapeutics, and 'tailored' compounds classified as doping agents according to the regulations of the World Anti-Doping Agency, with particular attention to analytical strategies enabling their detection in human blood or urine. Among these compounds, low- and high-molecular mass substances of peptidic (e.g. modified insulin-like growth factor-1, TB-500, hematide/peginesatide, growth hormone releasing peptides, AOD-9604, etc.) and non-peptidic (selective androgen receptor modulators, hypoxia-inducible factor stabilizers, siRNA, S-107 and ARM036/aladorian, etc.) as well as inorganic (cobalt) nature are considered and discussed in terms of specific requirements originating from physicochemical properties, concentration levels, metabolism, and their amenability for chromatographic-mass spectrometric or alternative detection methods.
The escalating problem of obesity has initiated an unprecedented search for novel anti-obesity drugs. This article reviews the wealth of central and peripheral targets that have been discovered recently. Some of the newer agents appear to have improved efficacy over current drugs, whereas others also have potential to treat the co-morbid risk factors associated with obesity. Future strategies are likely to combine drugs with complementary central and peripheral mechanisms of action to enhance therapeutic benefit.
Obesity, as in every western country, is currently the most prevalent chronic disease in childhood in Spain. This has led to obesity being one of the most common consultations in general paediatrics and, particularly, in paediatric endocrinology. Furthermore, obesity associated comorbidities are increasing in prevalence in children and adolescents. It is widely accepted that this increase in the prevalence of obesity is derived from an imbalance between energy intake and expenditure, associated to the lifestyle in western countries. However, there is increasing evidence of the role of individual and familial genetic background in the risk of developing obesity. The pathophysiological basis of the mechanisms responsible for the control of appetite and energy expenditure are being discovered on the basis of the increasing known cases of human monogenic, syndromic and endocrine obesity. Thus it is no longer appropiate to talk about obesity but rather about «obesities», as their pathophysiological bases differ and they require different diagnostic and management approaches.In 2011, the paediatrician must be aware of this issue and focus the clinical history and physical examination towards these specific clinical sign and symptoms, to better manage the available diagnostic and therapeutic resources when faced with a child with obesity.
Both human GH (hGH) and a lipolytic fragment (AOD9604) synthesized from its C-terminus are capable of inducing weight loss and increasing lipolytic sensitivity following longterm treatment in mice. One mechanism by which this may occur is through an interaction with the beta -adrenergic pathway, particularly with the beta (3)-adrenergic receptors (beta (3)-AR). Here we describe how hGH and AOD9604 can reduce body weight and body fat in obese mice following 14 d of chronic ip administration. These results correlate with increases in the level of expression of beta (3)-AR RNA, the major lipolytic receptor found In fat cells. Importantly, both hGH and AOD9604 are capable of increasing the repressed levels of beta (3)-AR RNA in obese mice to levels comparable with those in lean mice. The importance of beta (3)-AR was verified when long-term treatment with hGH and AOD9604 in beta (3)-AR knock-out mice failed to produce the change in body weight and increase in lipolysis that was observed in wild-type control mice. However, in an acute experiment, AOD9604 was capable of increasing energy expenditure and fat oxidation in the beta (3)-AR knock-out mice. In conclusion, this study demonstrates that the lipolytic actions of both hGH and AOD9604 are not mediated directly through the beta (3)-AR although both compounds increase beta (3)-AR expression, which may subsequently contribute to enhanced lipolytic sensitivity.
Available anti-obesity pharmacotherapy options remain very limited. The development of more effective drugs has become a priority. The potential strategies to achieve weight loss are to reduce energy intake, by stimulating anorexigenic signals or by blocking orexigenic signals, and to increase energy expenditure. This review will focus on approved obesity medications, as well as potential new pharmacological treatment options.
Experimental and clinical studies suggest that high serum levels of growth hormone (GH) increase cortical but not trabecular bone. We studied body composition and bone structure in transgenic mice (MT-bGH) with systemic overexpression of GH. Body composition was examined with dual-energy X-ray absorptiometry (DXA), ashing, and chemical analysis, and the femora with DXA and micro computerized tomography. The absolute fat and bone tissue contents were significantly higher in GH transgenic mice vs controls (P < or = 0.05), but no significant difference was noted when normalizing the values to body weight. Male transgenics displayed no change in apparent (volumetric) femoral bone density, relative cortical area and trabecular bone volume fraction. Female transgenic mice demonstrated an increase in apparent femoral density and in trabecular bone volume fraction (+130%; P < or = 0.01). The mineralized tissue matrix density was decreased in male and female transgenic mice (P < or = 0.05). The results show that chronic GH excess affects trabecular bone in a gender-specific manner and that bone changes depend on the compartment investigated.
Lipid storage and breakdown is mainly controlled by lipoprotein lipase and hormone-sensitive lipase. The aim of this work was to elucidate whether growth hormone mediated loss of adipose tissue involves a concerted action on tissue lipases, and to what degree such events are modulated by dietary regimen. Twelve-month-old rats fed first a high-fat diet or a low-fat diet for 14 weeks were injected with saline or growth hormone (4 mg/kg/d) for four days or three weeks in different combinations with either high- or low-fat diets. In adipose tissue, growth hormone generally inhibited lipoprotein lipase and also attenuated the inhibiting effect of insulin on hormone-sensitive lipase activity. Growth hormone treatment combined with restricted high-fat feeding reduced the activity of both lipases in adipose tissue and stimulated hormone-sensitive lipase in muscle. Generally, plasma levels of free fatty acids, glycerol and cholesterol were reduced by growth hormone, and in combination with restricted high-fat feeding, triglyceride levels improved too. We conclude that growth hormone inhibits lipid storage in adipose tissue by reducing both lipoprotein lipase activity and insulin's inhibitory action on hormone-sensitive lipase. We also propose that growth hormone's effects on tissue lipases and blood lipids are modulated by dietary regimen.
Obesity is a major health problem and as a result, it is reasonable to consider pharmacological approaches alongside approaches involving diet, physical activity and lifestyle change. The currently available drugs, sibutramine and orlistat, result in modest, clinically worthwhile weight loss, with demonstrable improvements in co-morbidity, but do not meet the often unrealistic expectations of patients or health care professionals managing obese patients. There is insufficient data on efficacy or safety of other agents to support their use. Many new pharmacological approaches are under investigation. These include gut hormones, such a peptide YY (3-36) and cholecystokinin that normally signal satiety, and centrally-acting agents such as serotonin agonists, the anticonvulsants topiramate and zonisamide, cannabinoid receptor antagonists and drugs that act on other peptide neurotransmitter systems such as NPY and the melanocortins. Given the multiple pathways that influence energy balance, it is likely that therapies targeting more than one control system may be required in the future to meet the expectations and needs of patients needing to lose weight for medical reasons.
Metabolic is developing AOD-9604 for the potential treatment of obesity. By February 2002, phase IIa trials were underway.
A number of anti-obesity drugs are currently undergoing clinical development. These include: (i) centrally-acting drugs, such as the noradrenergic and dopaminergic reuptake inhibitor radafaxine, the endocannabinoid antagonist rimonabant, the selective serotonin 5-HT2c agonist APD-356, and oleoyl-estrone; (ii) drugs that target peripheral episodic satiety signals, such as glucagon-like peptide-1 (exenatide, exenatide-LAR and liraglutide), peptide YY (intranasal PYY3-36 and AC-162325) and amylin (pramlintide); (iii) drugs that block fat absorption, such as the novel lipase inhibitors cetilistat and GT-389255; and (iv) a human growth hormone fragment (AOD-9604) that increases adipose tissue breakdown. Of these, only rimonabant has got as far as completing phase III clinical trials. This review will provide an overview of the most prominent drugs currently undergoing clinical development as potential anti-obesity therapies.
We have selectively synthesized a number of peptides encompassing the region of helix 3 of growth hormone (GH). These peptides and native human (h) GH have been evaluated for mitogenic and receptor activities in 3T3-F442A preadipocytes. In this system, wild type hGH is anti-mitogenic. In contrast, hGH 108-129 stimulated DNA synthesis while other GH-derived peptides were ineffective. hGH (L) 108-129 had an EC50 of about 0.2 nM and was maximally effective at about 0.5 nM in stimulating [3H]thymidine incorporation in 3T3-F442A cells. hGH (L) 108-129 was mitogenically as active as insulin-like growth factor-I and more active than insulin. It was less effective than transforming growth factor-beta. By cell cycle analysis, hGH (L) 108-129 increased the proportion of cells in S/G2/M phases to 28%. hGH, when coincubated with hGH (L) 108-129, blocked the mitogenic response of the peptide. A monoclonal antibody to the GH receptor significantly reduced binding of 125I-hGH to its receptor but had no effect on binding of 125I-hGH (L) 108-129. Affinity cross-linking of 125I-hGH to its receptor was not duplicated with 125I-hGH (L) 108-129. No other GH peptides or insulin competed for binding of 125I-hGH 108-129. Scatchard analysis indicated a Kd of 5.2 nM with 5.6 x 10(5) binding sites/cell for hGH (L) 108-129. These studies indicate that hGH (L) 108-129, a sequence encompassing helix 3 of hGH, acts by binding to a site other than the GH receptor and evokes high mitogenic responses.
Human GH represents a family of proteins rather than a single hormone. The circulation contains a bewildering array of GH forms, including several monomeric variants, their homo- and heteropolymers, fragments, and complexes with at least two BPs. The net biological activity of this mixture is difficult to predict, as the various molecular forms interact as partial agonists and/or antagonists at the receptor level. The number of GH forms that can be counted in plasma exceeds 100. Table 5 attempts to illustrate what is known and provide estimates for circulating variants. It does not include GH-V and its variants, which have to be added in pregnancy. Of note, what is commonly understood as "plasma GH," i.e. free monomeric 22K, represents only 21% of total immunoreactivity in plasma. In view of this complicated picture, it should be no surprise that different assays of plasma GH yield different results (107, 108, 290). While immunoassays are relatively unaffected by the BPs (291), receptor assays are seriously affected by the high affinity BP (261). Immunoassays, particularly of the monoclonal variety, are vulnerable to differential recognition of molecular variants depending on the unique epitope specificity of the antibody used. Polyclonal assays are more robust in this regard because of "epitope averaging" among the wide spectrum of epitope specificities present in the antibody population. Future work should aim at developing antibodies that are specific for individual GH variants. Such molecular probes will be helpful not only in standardizing immunoassays, but also in delineating the biological role of the various GH forms. The physiological significance of the numerous GH forms (or of the BPs) is still largely unknown. Progress in this area has been hampered, on the one hand, by the unavailability of pure GH variants in quantities sufficient for biological studies, and, on the other, by a certain lack of interest stemming from suspicions about artifacts. The recent resurgence of interest in GH and in its receptor and BPs should also refocus attention on the various molecular forms. Thus far, this interest has been largely confined to monomeric 22K, which is certainly effective for its original intended purpose, namely growth promotion. Whether 22K is sufficient for optimal growth and development, or whether it can fulfill all the functions of the GH family is unknown. It can be argued that evolutionarily conserved GH variants probably have biological importance.(ABSTRACT TRUNCATED AT 400 WORDS)
Normal postnatal somatic growth becomes progressively dependent on GH with time. In contrast to other hormones, GH is the only hormone known to produce a dose-dependent stimulation of postnatal growth. Most of the effects attributed to GH action appear to be the result of a direct effect of GH on cells in different peripheral tissues, including cartilage. In addition to the growth-stimulating effect, GH has the intrinsic properties of being able to exert both insulin-like and insulin-antagonistic effects in adipose tissue and skeletal muscle. These two apparently antagonistic effects seem to be explained by the stage of responsiveness of the target cells to GH, which is determined by the previous influence of endogenous GH. An inhibition of adenylate cyclase with a concomitant decrease in intracellular cAMP might be an important early cellular event in the course of GH action, but it is not known whether or how this change in nucleotide metabolism relates to the various expressed effects of the hormone. The recognition that GH directly interacts with chondrocytes in cartilage suggests that alterations in the concentration of circulating somatomedins cannot be the only factor regulating skeletal growth. The recent discovery by Green and coworkers (42) demonstrating that GH specifically stimulates the differentiation of cloned preadipose cells and myoblasts in tissue culture may be a major breakthrough in the understanding of the mechanism of action of the growth-promoting effect of GH. Green (42) has proposed that GH directly stimulates terminal differentiation of cells in many different tissues including epiphyseal plate cartilage. The finding that GH binds specifically to cells in the resting cell zone but not to differentiated chondrocytes in the growth plate suggests that prechondrocytes in the growth plate are the target cells for GH action. If it is correct that GH directly stimulates the differentiation of prechondrocytes, we suggest that, during the process of chondrocyte differentiation in the growth plate, the genes that code for growth factors of the somatomedin class, such as IGF-I, are expressed. As a consequence, the clonal expansion of the chondrocytes in the proliferative zone of the growth plate that occurs in vivo during the process of normal growth is the result of this local production of growth factors.
Our aim was to develop the glucose clamp (GC) technique in the conscious rat for assessment of in vivo insulin sensitivity. A 2-h euglycemic GC could be performed in chronically cannulated rats using 625 microliter blood. Overnight-fasted rats were infused with porcine insulin (1.67 mU . kg-1 . h-1). Insulin levels of 41 +/- 2 (SE) mU/liter were produced in rats aged 91 +/- 4 days with a 60- to 120-min glucose infusion rate (GIR60-120) of 10.6 +/- 0.6 mg . kg-1 . min-1 (n = 9) during euglycemia. GIR60-120 was significantly (P less than 0.025) reduced in rats aged greater than 130 days (mean, 169 +/- 16 days) to 7.7 +/- 1.2 mg . kg-1 . min-1 (n = 7). Metabolic clearance rate of porcine insulin (46 +/- 3 ml . kg-1 . min-1) and GIR60-120 compared with plateau plasma insulin levels are higher than values reported in humans. The latter may be due to suppression of a higher basal hepatic glucose production or increased potency of porcine compared with native insulin. We conclude that the GC can be accomplished in the rat. When combined with tracer administration and subsequent killing, it should provide a quantitative in vivo measurement of insulin sensitivity in individual tissues.
Various bovine growth hormone (GH) fragments were prepared and tested for somatomedin-like activity in vitro. Cyanogen bromide cleavage followed by reduction and alkylation yielded three fragments which were identified as GH (6-124), GH (150-179) and GH (125-149). No consistent effect was found when these preparations were tested for their ability to stimulate in vitro sulfate and thymidine uptake by rat costal cartilage and to compete with [125I]iodoinsulin for insulin-binding sites on placenta membranes. A fourth peptide was isolated by cleavage of the tryptophanyl and methionly bonds of bovine Gh using anhydrous heptofluorobutyric acid and cyanogen bromide. In addition to significant amounts of non-specific cleavage products, a peptide have a molecular weight of about 4800 was isolated. The amino terminal residue was leucine and the carboxyl terminal was homoserine. These data, in addition to the amino acid composition, suggested that the peptide corresponded to residues 87-124. Fragment GH (87-124) stimulated sulfate (minimum effective concentration, 5 . 10-8 M) and thymidine (minimum effective concentration, 10-8M) uptake by rat costal cartilage. It also cross-reacted, albeit weakly, with insulin-binding sites on placenta membrane. Maximum displacement was 35% of non-specific binding. These observations demonstrate that somatomedin-like activity can be generated from the growth hormone molecule which is inherently devoid of such activity.
We conducted a controlled trial of recombinant human GH (rhGH) in 27 healthy elderly women (66.7 +/- 3.0 yr), of whom 8 took a stable dose of replacement estrogen throughout the study (plus estrogen group). Hormone or placebo was given as a single daily injection. A total of 19 women were assigned to receive rhGH at an initial daily dose of 0.043 mg/kg BW. After several weeks, 50% dose reductions were necessitated by side-effects. The last 7 subjects to be enrolled began treatment at this reduced level. A total of 13 women assigned to rhGH and 14 women assigned to placebo completed 6 months of drug treatment. In the rhGH group, 6 women took estrogen; thus, the effects of rhGH were assessed separately by estrogen status. Circulating insulin-like growth factor-I (IGF-I) levels were similar at baseline (rhGH, 133 +/- 40.4 micrograms/L; placebo, 128 +/- 13). rhGH increased IGF-I and IGF-I-binding protein-3 (IGFBP-3) in all subjects [6 month IGF-I in plus estrogen women, 230 +/- 25.4 micrograms/L; in those not receiving estrogen (minus estrogen), 308 +/- 21.3]. No changes in IGF-I or IGFBP-3 occurred with placebo (IGF-I, 144 +/- 21.3 micrograms/L). Skinfold thickness measurements showed an 11% decrease in fat mass (P < 0.005) and a 9% decrease in percent fat after 6 months of rhGH treatment. No significant difference in nitrogen balance was seen in either group at 6 months, but rhGH increased creatinine clearance by 9.2% (P < 0.05). rhGH dramatically increased markers of bone turnover, with more pronounced effects in minus estrogen women. Hydroxyproline excretion increased by 20% and 80%, and pyridinoline excretion increased by 44% and 75% in plus and minus estrogen subgroups, respectively. Osteocalcin concentrations increased by more than 60% in minus estrogen women (P < 0.05), but did not change in the plus estrogen group. No changes were observed in circulating type I procollagen extension peptide in either group, and no change in any turnover marker was seen in the placebo group. rhGH did not alter blood pressure or circulating L-T4 levels, but a transient increase in serum T3 was observed in the minus estrogen group at 3 months. rhGH decreased low density lipoprotein cholesterol in the minus estrogen group, but otherwise no significant changes in circulating lipoproteins or fibrinogen were observed. Eight women assigned to rhGH and 14 placebo-treated women remained on blinded treatment through 12 months.(ABSTRACT TRUNCATED AT 400 WORDS)
The effects of long-term treatment of C57BL/6J (ob/ob) mice with a synthetic carboxylterminal sequence of human growth hormone, hGH 177-191, were investigated. Results indicate that the hGH 177-191 reduced the cumulative body weight gain, and decreased the adipose tissue mass. The lipogenesis in adipose tissues was significantly inhibited by the treatment with hGH 177-191. These findings support the suggestion that hGH 177-191 is the functional domain of hGH for the antilipogenic actions of the intact hormone both in vivo and in vitro. The hGH 177-191 peptide has the potential to be an effective compound for the treatment of human obesity and for the improvement of meat qualities in farm animals.
The effect of a synthetic C-terminal peptide sequence of human growth hormone, Leu-Arg-Ile-Val-Gln-Cys-Arg-Val-Ser-Glu-Gly-Ser-Cys-Gly-Phe (hGH 177-191) on glucose transport in adipocytes isolated from genetically-obese Zucker rats was investigated. The results showed that the synthetic peptide induced a reduction of basal and insulin-stimulated D[1-14C]-2-deoxyglucose uptake in isolated adipocytes. In comparison with the intact molecule of human growth hormone (hGH), the synthetic peptide at equimolar concentrations was found to be more potent. These findings are consistent with the suggestion that the functional domain responsible for the antilipogenic activity of hGH resides in the C-terminal region of the hGH molecule and the effect on glucose transport may contribute, at least in part, to the antilipogenic property of the peptide hGH 177-191 as well as of the intact hormone.
The synthetic C-terminal peptide fragment of human growth hormone, Leu-Arg-Ile-Val Gln-Cys-Arg-Val-Ser-Glu-Gly-Ser-Cys-Gly-Phe (hGH 177-191), was shown to have antilipogenic activity identical with that of the intact molecule of human growth hormone (hGH). No significant lipolytic effect of hGH 177-191 was found as determined by the rate of glycerol release from epididymal fat pads of the peptide-treated rats. The results support the suggestion that the functional domain responsible for the antilipogenic activity of hGH resides in the C-terminal region of the molecule and that the main physiological effect of hGH in lipid metabolism is at the level of lipogenesis.
To determine whether growth hormone replacement in older men improves functional ability.
Randomized, controlled, double-blind trial.
General community.
52 healthy men older than 69 years of age with well-preserved functional ability but low baseline insulin-like growth factor 1 levels.
Growth hormone (0.03 mg/kg of body weight) or placebo given three times a week for 6 months.
Body composition, knee and hand grip muscle strength, systemic endurance, and cognitive function.
The participants' mean age was 75.0 years (range, 70 to 85 years). At 6 months, lean mass had increased on average by 4.3% in the growth hormone group and had decreased by 0.1% in the placebo group, a difference of 4.4 percentage points (95% CI, 2.1 to 6.8 percentage points). Fat mass decreased by an average of 13.1% in the growth hormone group and by 0.3% in the placebo group, a difference of 12.8 percentage points (CI, 8.6 to 17.0 percentage points). No statistically or clinically significant differences were seen between the groups in knee or hand grip strength or in systemic endurance. The mean Trails B score in the growth hormone group improved by 8.5 seconds, whereas scores in the placebo group deteriorated by 5.0 seconds, a difference of 13.5 seconds (CI, 3.1 seconds to 23.9 seconds; P = 0.01). However, the growth hormone group's score on the Mini-Mental Status Examination deteriorated by 0.4, whereas the placebo group's score improved by 0.2, a difference of 0.6 (P = 0.11). The two treatment groups had almost identical scores on the Digit Symbol Substitution Test (P > 0.2). Twenty-six men in the growth hormone group had 48 incidents of side effects, and 26 placebo recipients had 14 incidents of side effects (P = 0.002). Dose reduction was required in 26% of the growth hormone recipients and in none of the placebo recipients (P < 0.001).
Physiologic doses of growth hormone given for 6 months to healthy older men with well-preserved functional abilities increased lean tissue mass and decreased fat mass. Although body composition improved with growth hormone use, functional ability did not improve. Side effects occurred frequently.
The secretion of both growth hormone (GH) and androgens declines with age which may play a role in the senescent changes in body composition and organ function. Among healthy adults abdominal adiposity is an important negative determinant of GH secretion. Surprisingly, abdominal or android obesity seems inversely correlated with testosterone levels in males but not in females. The ability of GH to promote lipolysis and preserve or increase lean body mass has been reappraised in substitution studies in GH-deficient adults. By comparison, adequately controlled studies of androgen replacement in hypogonadal and/or elderly males are few. In view of the physiological and clinical relevance of obtaining information about the aging process, there is a need for controlled experiments addressing similarities and differences between the action of GH and sex steroids in adults.
A range of peptide analogues related to Asu11-human growth hormone (6-13) have been synthesized and tested for hypoglycaemic activity using in vivo insulin tolerance tests. Utilising an alanine scan procedure and a selective amino acid residue approach these structure-activity studies suggest that residues Phe10, Arg8 and the C-terminal beta-turn structure are important for the expression of biological activity.
The major isoform of hGH is a polypeptide of 191 amino acids. Human GH1-43 is an amino terminal segment of hGH1-191 which comprises the first 43 amino acids. This peptide is a potent regulator of glucose homeostasis. To facilitate our understanding of the physiological regulation of hGH1-43 an assay to measure its levels in biological fluids and extracts is needed. This communication describes the development of anti-hGH1-43 monoclonal antibodies and their use in the development of an indirect competitive ELISA for the quantification of hGH1-43. Hybridomas were produced by the fusion of FOX-NY myeloma cells with spleen cells taken from a mouse immunized with hGH1-43. The hybridomas were screened for production of antibodies to hGH1-43 by antibody capture ELISA. Hybridomas which produced antibodies reactive to hGH1-43 were cloned by limiting dilution. Three monoclonal hybridomas, CCL-1, CCL-2, and CCL-3 were subsequently obtained. These hybridomas secreted antibodies that were highly reactive towards hGH1-43 but minimally reactive towards hGH1-191. The isotypes of the mAbs secreted by CCL-1, CCL-2 and CCL-3 were all IgG1 kappa as shown by isotype specific antibody capture analysis. An indirect competitive ELISA with a detection limit that ranged from 1 to 10 ng/ml was developed using mAbs from monoclonal hybridoma CCL-3. Dose-response curves for competing hGH1-191, hPRL, and hPL indicated minimal cross-reactivity of mAbs with these hormones and conversely, a high degree of specificity for hGH1-43. Dose-response curves for dilutions of human serum and pituitary extract were parallel to the standard. The availability of a sensitive assay for the measurement of hGH1-43 will help us answer questions regarding the biosynthesis, regulation of secretion, and role of hGH1-43 in the control of glucose homeostasis.
