Using Target Engagement Biomarkers to Predict Clinical Efficacy of MetAP2 Inhibitors.

Target-engagement pharmacodynamic (PD) biomarkers are valuable tools in the prioritization of drug candidates, especially for novel, first-in-class mechanisms whose robustness to alter disease outcome is unknown. Methionine aminopeptidase 2 (MetAP2) is a cytosolic metalloenzyme that cleaves the N-terminal methionine from nascent proteins. Inhibition of MetAP2 leads to weight loss in obese rodents, dogs and humans. However, there is a need to develop efficacious compounds that specifically inhibit MetAP2 with an improved safety profile. The objective of this study was to identify a PD biomarker for selecting potent, efficacious compounds and for predicting clinical efficacy that would result from inhibition of MetAP2. Here we report the use of NMet14-3-3γ for this purpose. Treatment of primary human cells with MetAP2 inhibitors resulted in an approx. 10-fold increase in NMet14-3-3γ levels. Furthermore, treatment of diet-induced obese mice with these compounds reduced body weight (approx. 20%) and increased NMet14-3-3γ (approx. 15-fold) in adipose tissues. The effects on target engagement and body weight increased over time and were dependent on dose and administration frequency of compound. The relationship between compound concentration in plasma, NMet14-3-3γ in tissue, and reduction of body weight in obese mice was used to generate a pharmacokinetic-pharmacodynamic-efficacy model for predicting efficacy of MetAP2 inhibitors in mice. We also developed a model for predicting weight loss in humans using a target engagement PD assay that measures inhibitor-bound MetAP2 in blood. In summary, MetAP2 target engagement biomarkers can be used to select efficacious compounds and predict weight loss in humans. SIGNIFICANCE STATEMENT: The application of target engagement pharmacodynamic biomarkers during drug development provides a means to determine the dose required to fully engage the intended target and an approach to connect the drug target to physiological effects. This work exemplifies the process of using target engagement biomarkers during preclinical research to select new drug candidates and predict clinical efficacy. We determine concentration of MetAP2 antiobesity compounds needed to produce pharmacological activity in primary human cells and in target tissues from an appropriate animal model and establish key relationships between pharmacokinetics, pharmacodynamics, and efficacy, including the duration of effects after drug administration. The biomarkers described here can aid decision-making in early clinical trials of MetAP2 inhibitors for the treatment of obesity.

MetAP2 inhibition increases energy expenditure through direct action on brown adipocytes.

Inhibitors of methionine aminopeptidase 2 (MetAP2) have been shown to reduce body weight in obese mice and humans. The target tissue and cellular mechanism of MetAP2 inhibitors, however, have not been extensively examined. Using compounds with diverse chemical scaffolds, we showed that MetAP2 inhibition decreases body weight and fat mass and increases lean mass in the obese mice but not in the lean mice. Obesity is associated with catecholamine resistance and blunted ß-adrenergic receptor signaling activities, which could dampen lipolysis and energy expenditure resulting in weight gain. In the current study, we examined effect of MetAP2 inhibition on brown adipose tissue and brown adipocytes. Norepinephrine increases energy expenditure in brown adipose tissue by providing fatty acid substrate through lipolysis and by increasing expression of uncoupled protein-1 (UCP1). Metabolomic analysis shows that in response to MetAP2 inhibitor treatment, fatty acid metabolites in brown adipose tissue increase transiently and subsequently decrease to basal or below basal levels, suggesting an effect on fatty acid metabolism in this tissue. Treatment of brown adipocytes with MetAP2 inhibitors enhances norepinephrine-induced lipolysis and energy expenditure, and prolongs the activity of norepinephrine to increase ucp1 gene expression and energy expenditure in norepinephrine-desensitized brown adipocytes. In summary, we showed that the anti-obesity activity of MetAP2 inhibitors can be mediated, at least in part, through direct action on brown adipocytes by enhancing ß-adrenergic-signaling-stimulated activities.

CX3CL1-Fc treatment prevents atherosclerosis in Ldlr KO mice.

OBJECTIVE: Atherosclerosis is a major cause of cardiovascular disease. Monocyte-endothelial cell interactions are partly mediated by expression of monocyte CX3CR1 and endothelial cell fractalkine (CX3CL1). Interrupting the interaction between this ligand-receptor pair should reduce monocyte binding to the endothelial wall and reduce atherosclerosis. We sought to reduce atherosclerosis by preventing monocyte-endothelial cell interactions through use of a long-acting CX3CR1 agonist. METHODS: In this study, the chemokine domain of CX3CL1 was fused to the mouse Fc region to generate a long-acting soluble form of CX3CL1 suitable for chronic studies. CX3CL1-Fc or saline was injected twice a week (30 mg/kg) for 4 months into Ldlr knockout (KO) mice on an atherogenic western diet. RESULTS: CX3CL1-Fc-treated Ldlr KO mice showed decreased en face aortic lesion surface area and reduced aortic root lesion size with decreased necrotic core area. Flow cytometry analyses of CX3CL1-Fc-treated aortic wall cell digests revealed a decrease in M1-like polarized macrophages and T cells. Moreover, CX3CL1-Fc administration reduced diet-induced atherosclerosis after switching from an atherogenic to a normal chow diet. In vitro monocyte adhesion studies revealed that CX3CL1-Fc treatment caused fewer monocytes to adhere to a human umbilical vein endothelial cell monolayer. Furthermore, a dorsal window chamber model demonstrated that CX3CL1-Fc treatment decreased in vivo leukocyte adhesion and rolling in live capillaries after short-term ischemia-reperfusion. CONCLUSION: These results indicate that CX3CL1-Fc can inhibit monocyte/endothelial cell adhesion as well as reduce atherosclerosis.

Chronic fractalkine administration improves glucose tolerance and pancreatic endocrine function.

We have previously reported that the fractalkine (FKN)/CX3CR1 system represents a novel regulatory mechanism for insulin secretion and ß cell function. Here, we demonstrate that chronic administration of a long-acting form of FKN, FKN-Fc, can exert durable effects to improve glucose tolerance with increased glucose-stimulated insulin secretion and decreased ß cell apoptosis in obese rodent models. Unexpectedly, chronic FKN-Fc administration also led to decreased α cell glucagon secretion. In islet cells, FKN inhibited ATP-sensitive potassium channel conductance by an ERK-dependent mechanism, which triggered ß cell action potential (AP) firing and decreased α cell AP amplitude. This results in increased glucose-stimulated insulin secretion and decreased glucagon secretion. Beyond its islet effects, FKN-Fc also exerted peripheral effects to enhance hepatic insulin sensitivity due to inhibition of glucagon action. In hepatocytes, FKN treatment reduced glucagon-stimulated cAMP production and CREB phosphorylation in a pertussis toxin-sensitive manner. Together, these results raise the possibility of use of FKN-based therapy to improve type 2 diabetes by increasing both insulin secretion and insulin sensitivity.

Time-dependent inhibition of PHD2.

Prolyl hydroxylases (PHDs) down-regulate the level of hypoxia-inducible factors (HIFs) by hydroxylating key proline residues that trigger the degradation of the protein and affect the cell and its ability to respond to hypoxic stress. Several small molecule PHD inhibitors are now in various preclinical and clinical stages for the treatment of anemia. The present study provides a detail kinetic analysis for some of these inhibitors. The data generated in the present study suggest that these compounds are reversible and compete directly with the co-substrate, 2-oxoglutarate (2-OG) for binding at the enzyme active site. Most of these compounds are pan PHD inhibitors and exhibit a time-dependent inhibition (TDI) mechanism due to an extremely slow dissociation rate constant, koff, and a long residence time.

Discovery of Highly Potent Liver X Receptor ß Agonists.

Introducing a uniquely substituted phenyl sulfone into a series of biphenyl imidazole liver X receptor (LXR) agonists afforded a dramatic potency improvement for induction of ATP binding cassette transporters, ABCA1 and ABCG1, in human whole blood. The agonist series demonstrated robust LXRß activity (>70%) with low partial LXRα agonist activity (<25%) in cell assays, providing a window between desired blood cell ABCG1 gene induction in cynomolgus monkeys and modest elevation of plasma triglycerides for agonist 15. The addition of polarity to the phenyl sulfone also reduced binding to the plasma protein, human α-1-acid glycoprotein. Agonist 15 was selected for clinical development based on the favorable combination of in vitro properties, excellent pharmacokinetic parameters, and a favorable lipid profile.

Discovery of potent, reversible MetAP2 inhibitors via fragment based drug discovery and structure based drug design-Part 2.

Methionine aminopeptidase-2 (MetAP2) is an enzyme that cleaves an N-terminal methionine residue from a number of newly synthesized proteins. This step is required before they will fold or function correctly. Pre-clinical and clinical studies with a MetAP2 inhibitor suggest that they could be used as a novel treatment for obesity. Herein we describe the discovery of a series of pyrazolo[4,3-b]indoles as reversible MetAP2 inhibitors. A fragment-based drug discovery (FBDD) approach was used, beginning with the screening of fragment libraries to generate hits with high ligand-efficiency (LE). An indazole core was selected for further elaboration, guided by structural information. SAR from the indazole series led to the design of a pyrazolo[4,3-b]indole core and accelerated knowledge-based fragment growth resulted in potent and efficient MetAP2 inhibitors, which have shown robust and sustainable body weight loss in DIO mice when dosed orally.

Discovery of potent, reversible MetAP2 inhibitors via fragment based drug discovery and structure based drug design-Part 1.

Methionine aminopeptidase 2 (MetAP2) is an enzyme that cleaves an N-terminal methionine residue from a number of newly synthesized proteins. Pre-clinical and clinical studies suggest that MetAP2 inhibitors could be used as a novel treatment for obesity. Herein we describe our use of fragment screening methods and structural biology to quickly identify and elaborate an indazole fragment into a series of reversible MetAP2 inhibitors with <10nM potency, excellent selectivity, and favorable in vitro safety profiles.

The Selective PI3K Inhibitor XL147 (SAR245408) Inhibits Tumor Growth and Survival and Potentiates the Activity of Chemotherapeutic Agents in Preclinical Tumor Models.

Dysregulation of PI3K/PTEN pathway components, resulting in hyperactivated PI3K signaling, is frequently observed in various cancers and correlates with tumor growth and survival. Resistance to a variety of anticancer therapies, including receptor tyrosine kinase (RTK) inhibitors and chemotherapeutic agents, has been attributed to the absence or attenuation of downregulating signals along the PI3K/PTEN pathway. Thus, PI3K inhibitors have therapeutic potential as single agents and in combination with other therapies for a variety of cancer indications. XL147 (SAR245408) is a potent and highly selective inhibitor of class I PI3Ks (α, ß, γ, and δ). Moreover, broad kinase selectivity profiling of >130 protein kinases revealed that XL147 is highly selective for class I PI3Ks over other kinases. In cellular assays, XL147 inhibits the formation of PIP3 in the membrane, and inhibits phosphorylation of AKT, p70S6K, and S6 in multiple tumor cell lines with diverse genetic alterations affecting the PI3K pathway. In a panel of tumor cell lines, XL147 inhibits proliferation with a wide range of potencies, with evidence of an impact of genotype on sensitivity. In mouse xenograft models, oral administration of XL147 results in dose-dependent inhibition of phosphorylation of AKT, p70S6K, and S6 with a duration of action of at least 24 hours. Repeat-dose administration of XL147 results in significant tumor growth inhibition in multiple human xenograft models in nude mice. Administration of XL147 in combination with chemotherapeutic agents results in antitumor activity in xenograft models that is enhanced over that observed with the corresponding single agents.

Liver X receptor (LXR) partial agonists: biaryl pyrazoles and imidazoles displaying a preference for LXRß.

A series of biaryl pyrazole and imidazole Liver X Receptor (LXR) partial agonists has been synthesized displaying LXRß selectivity. The LXRß selective partial agonist 18 was identified with potent induction of ATP binding transporters ABCA1 and ABCG1 in human whole blood (EC50=1.2µM, 55% efficacy). In mice 18 displayed peripheral induction of ABCA1 at 3 and 10mpk doses with no significant elevation of plasma or hepatic triglycerides at these doses, showing an improved profile compared to a full pan-agonist.

Discovery and characterization of an inhibitor of glucosylceramide synthase.

Targeting glycosphingolipid synthesis has emerged as a novel approach for treating metabolic diseases. 32 (EXEL-0346) represents a new class of glucosylceramide synthase (GCS) inhibitors. This report details the elaboration of hit 8 with the goal of achieving and maintaining maximum GCS inhibition in vivo. 32 inhibited GCS with an IC(50) of 2 nM and achieved maximum hepatic GCS inhibition after four or five daily doses in rodents. Robust improvements in glucose tolerance in DIO mice and ZDF rats were observed after 2 weeks of q.d. dosing. Four weeks of dosing resulted in decreased plasma triglycerides and reduced hepatic fat deposition. Thus, 32 provides insight into the amount of metabolic regulation that can be restored following achievement of maximal target knockdown.

Inhibition of proliferation in human MNNG/HOS osteosarcoma and SK-ES-1 Ewing sarcoma cell lines in vitro and in vivo by antagonists of growth hormone-releasing hormone: effects on insulin-like growth factor II.

BACKGROUND: Antagonists of growth hormone-releasing hormone (GH-RH) can inhibit the proliferation of various tumors either indirectly through the suppression of the pituitary growth hormone/hepatic insulin-like growth factor I (IGF-I) axis and the lowering of serum IGF-I concentration or directly by reducing the levels of IGF-I and IGF-II and their mRNA expression in tumors and blocking the effect of autocrine GH-RH. In this study, the authors investigated the effects of the GH-RH antagonist JV-1-38 on MNNG/HOS human osteosarcoma and SK-ES-1 human Ewing sarcoma cell lines. METHODS: Male nude mice bearing subcutaneous xenografts of MNNG/HOS or SK-ES-1 tumors were treated subcutaneously with JV-1-38 at a dose of 20 microg twice daily for 4 weeks. The concentrations of IGF-I and IGF-II in serum and in tumor tissue were measured by radioimmunoassay. Tumor and liver levels of mRNA for IGF-I and IGF-II were determined by reverse transcriptase-polymerase chain reaction analysis. The effects of JV-1-38, IGF-I, and IGF-II on cell proliferation in vitro were evaluated. RESULTS: GH-RH antagonist significantly (P < 0.05) inhibited the tumor volume and tumor weight of MNNG/HOS and SK-ES-1 tumors by > 50% after 4 weeks and increased tumor doubling time. JV-1-38 lowered the serum IGF-I level, decreased the expression of mRNA for IGF-I in the liver, and significantly (P < 0.05-0.01) reduced the concentration of IGF-II and mRNA levels for IGF-II in both sarcomas. The concentration of IGF-I was lowered only in SK-ES-1 tumors. In vitro, the proliferation of SK-ES-1 and MNNG/HOS cells was inhibited by JV-1-38 and by antisera to IGF-I and IGF-II. CONCLUSIONS: The inhibition of MNNG/HOS osteosarcoma and SK-ES-1 Ewing sarcoma by GH-RH antagonists was linked to a suppression of IGF-II production in tumors. However, in SK-ES-1 tumors, the effects on IGF-I also may be involved.

Inhibition of proliferation of PC-3 human prostate cancer by antagonists of growth hormone-releasing hormone: lack of correlation with the levels of serum IGF-I and expression of tumoral IGF-II and vascular endothelial growth factor.

BACKGROUND: Antagonists of growth hormone-releasing hormone (GHRH) such as JV-1-38 can inhibit androgen-independent prostate cancer directly by several mechanisms and/or indirectly by suppressing growth hormone/insulin-like growth factor-I (GH/IGF-I) axis. To shed more light on the mechanisms involved, the effects of JV-1-38 on PC-3 human prostate cancer were compared with those of somatostatin analog RC-160 in vivo and in vitro. METHODS: Nude mice bearing PC-3 tumors received JV-1-38 (20 microg), RC-160 (50 microg) or a combination of JV-1-38 and RC-160. The concentration of IGF-I in serum and the expression of mRNA for IGF-II and vascular endothelial growth factor (VEGF) in tumor tissue were investigated. RESULTS: In vivo, the final volume of PC-3 tumors treated with JV-1-38 was significantly lowered by 49% (P < 0.01), whereas RC-160 exerted only 30% inhibition (NS), compared with controls. Combined use of both compounds augmented tumor inhibition to 63% (P < 0.001). Serum IGF-I levels were decreased only in mice treated with RC-160. JV-1-38 suppressed mRNA for IGF-II in PC-3 tumors by 42%, whereas RC-160 alone or in combination with JV-1-38 caused a 65% reduction. JV-1-38 and RC-160 used as single drugs decreased the expression of VEGF by 50%, and their combination caused a 63% reduction. In vitro, JV-1-38 inhibited the proliferation of PC-3 cells by 39%. This effect could be partially reversed by addition of IGF-I to the serum-free medium. RC-160 alone did not affect the PC-3 cell growth in vitro, but in combination with JV-1-38 it augmented the antiproliferative effect of the GH-RH antagonist to 72%. Exposure to JV-1-38 in vitro reduced the expression of mRNA for IGF-II in PC-3 cells by 55% but did not change VEGF mRNA levels, whereas RC-160 had no effect. CONCLUSIONS: The antiproliferative effect of JV-1-38 was not associated with the suppression of serum IGF-I and was only partially correlated with the expression of IGF-II and VEGF in PC-3 tumors, suggesting that other mechanisms play a role in the antitumor action of GHRH antagonists. Nevertheless, the stronger inhibition of tumor growth after combined treatment with JV-1-38 and RC-160 indicates that the interference with multiple local stimulatory factors leads to an enhanced inhibition of prostate cancer.

Expression of growth hormone-releasing hormone (GHRH) and splice variants of GHRH receptors in human experimental prostate cancers.

The expression of mRNA for GHRH and splice variants (SVs) of GHRH receptors in LNCaP, MDA-PCa-2b and PC-3 human prostate cancers grown in nude mice was investigated by RT-PCR. The expression of mRNA for GHRH was detected in LNCaP and PC-3, but not in MDA-PCa-2b prostatic carcinoma. RT-PCR analyses of mRNA isolated from LNCaP, MDA-PCa-2b and PC-3 cancers, revealed the presence of 720 and 566 bp products, corresponding to SV(1) and SV(2) isoforms of GHRH receptors. In PC-3 tumor membranes a radiolabeled GHRH antagonist [125I]-JV-1-42 was bound to one class of high-affinity binding sites (K(d)=1.81+/-0.47 nM) and maximum binding capacity of 332.7+/-27.8 fmol/mg membrane protein. The in vivo uptake of [125I]-JV-1-42 was observed in all xenografts of human prostate cancer, the tracer accumulation being the highest in PC-3 tumors. These results indicate that GHRH and SVs of its receptors, different from those found in the pituitary, are present in experimental human prostate cancers and may form a local mitogenic loop. The antiproliferative effects of GHRH antagonists on growth of prostate cancer could be exerted in part by an interference with this local GHRH system.

Inhibition of PC-3 human prostate cancers by analogs of growth hormone-releasing hormone (GH-RH) endowed with vasoactive intestinal peptide (VIP) antagonistic activity.

Vasoactive intestinal peptide (VIP) stimulates the proliferation and invasiveness of malignant prostatic cells. Receptors for VIP and the closely related growth hormone-releasing hormone (GH-RH) show considerable homology and are found in prostatic and other carcinomas. Among various analogs of GH-RH synthesized, JV-1-52 is a non-selective VIP/GH-RH antagonist, whereas JV-1-53 is a VIP antagonist devoid of GH-RH antagonistic effect. In our study, nude mice bearing PC-3 human androgen-independent prostate carcinomas were treated with JV-1-52 or JV-1-53 (20 microg/day, s.c.) for 28 days. Both antagonists produced a similar reduction in tumor volume (62-67%, p < 0.01) and tumor weight (59-62%; p < 0.05) vs. controls and extended tumor doubling-time from 9.1 to about 16 days (p < 0.05). To investigate the mechanisms involved, in another study we compared the effects of JV-1-53 with those of somatostatin analog RC-160. VIP antagonist JV-1-53 reduced tumor weight by 67% (p < 0.01) and suppressed the expression of mRNA for c-fos and c-jun oncogenes by about 34% (p < 0.05), without affecting serum levels of insulin-like growth factor-I (IGF-I). In contrast, RC-160 (50 microg/day) reduced serum IGF-I by 19% (p < 0.05), but did not significantly decrease tumor weight. mRNA for VIP and high affinity receptors for VIP were detected on PC-3 tumors. Our results suggest that VIP/GH-RH antagonists can inhibit the growth of androgen-independent prostate cancer by abrogating the autocrine/paracrine mitogenic stimuli of VIP. The ability of GH-RH antagonists to block tumoral VIP receptors, in addition to GH-RH receptors, could be potentially beneficial for prostate cancer therapy.

Effective treatment of experimental DU-145 prostate cancers with targeted cytotoxic somatostatin analog AN-238.

We evaluated the effectiveness of targeted cytotoxic analog of somatostatin (SST) AN-238, consisting of 2-pyrrolinodoxorubicin (AN-201) linked covalently to SST octapeptide carrier RC-121 in DU-145 human androgen-independent prostate cancers xenografted into nude mice. We also investigated the expression of mRNAs for SST receptor subtypes 2A and 5 (sst2A and sst5) in DU-145 tumors. After 8 weeks of treatment, AN-238 practically arrested the proliferation of DU-145 cancers. The tumor volume in nude mice that received 4 injections of AN-238 at the dose of 150 nmol/kg was 63.4+/-6.7 mm3, nearly 4 times smaller than that in controls which measured 249.1+/-36.3 mm3 (p<0.001). Treatment with AN-238 lowered tumor weight by 68% (p<0.01) compared with the control group and extended the tumor volume doubling time to 184.1+/-69.4 days, versus 32.1+/-6.6 days in controls (p<0.05). No toxicity-related deaths occurred during treatment with AN-238. Cytotoxic radical AN-201 administered alone or in an unconjugated mixture with carrier RC-121 inhibited the growth of DU-145 tumors only after the third and fourth injection and was toxic. The expression of mRNA for sst2A and sst5 was detected in all specimens of control DU-145 tumors and in tumors treated with AN-238. The present study demonstrates the high efficacy of SST-receptor-targeted chemotherapy in a model of human androgen-independent prostatic carcinoma.

Inhibition of in vivo proliferation of MDA-PCa-2b human prostate cancer by a targeted cytotoxic analog of luteinizing hormone-releasing hormone AN-207.

The efficacy of therapy with targeted cytotoxic luteinizing hormone-releasing hormone (LHRH) analog AN-207 consisting of superactive doxorubicin derivative AN-201 linked to carrier [D-Lys(6)]LH-RH was evaluated in vivo in nude mice bearing xenografts of MDA-PCa-2b prostate cancer line. AN-207 was administered intravenously (i.v.) at 200 nmol/kg on day 1 and at 150 nmol/kg on day 14. After 4 weeks of treatment with AN-207, tumor growth was inhibited as shown by a 63% (P<0.01) decrease in tumor volume and a 55% (P<0.05) reduction in tumor weight, compared with controls. None of the animals died after administration of AN-207 at the total dose of 350 nmol/kg, and at the end of the experiment the body weights of mice given AN-207 did not differ significantly from controls. A single injection of cytotoxic radical AN-201 at 200 nmol/kg resulted in 43% mortality. In the surviving mice, AN-201 caused a 50% inhibition in tumor volume and a 27% reduction in tumor weight, which were non-significant, as compared to the controls. After 4 weeks, serum prostate-specific antigen concentrations in mice treated with AN-207 were 65% lower than those in controls (P<0.05), while in animals given AN-201 the reduction in serum prostate-specific antigen was only 40% (NS). The expression of mRNA for LHRH receptors was detected by reverse transcriptase polymerase chain reaction (RT-PCR) in MDA-PCa-2b tumors. The present study indicates that chemotherapy targeted to LHRH receptors on tumors inhibits growth of MDA-PCa-2B prostate cancers representative of human carcinoma disseminated to the bone and progressing despite androgen withdrawal.

New analogs of somatostatin: inhibiting effectively GH, glucagon and insulin levels.

The in vivo effects of three new analogs of somatostatin (ASS-51, ASS-52 and ASS-53 analogs) on GH, insulin and glucagon were studied in WKY rats. The solid phase method was used for the synthesis of ASS. Octreotide and ASS were given iv. in a dose of 0.05 &mgr;g/kg per animal in a time-dependent manner. ASS-52 and ASS-53 were longer acting and more potent somatostatin analogs when compared to octreotide in producing the inhibition of GH. ASS-51 was found to be the most potent and selective inhibitor of insulin and glucagon release. Our results show that the increased inhibitory effect and the higher selectivity of the new somatostatin analogs may result from the differences in their chemical structure. ASS-52 is most active in inhibiting GH release. The mechanism by which ASS-52 inhibits preferentially GH release may involve the opioid system and the activation of GABA-ergic receptors. In studies in vitro ASS-52 inhibited GH release from pituitary cells" culture.