Metabolomic changes of the multi (-AGC-) kinase inhibitor AT13148 in cells, mice and patients are associated with NOS regulation.

INTRODUCTION: To generate biomarkers of target engagement or predictive response for multi-target drugs is challenging. One such compound is the multi-AGC kinase inhibitor AT13148. Metabolic signatures of selective signal transduction inhibitors identified in preclinical models have previously been confirmed in early clinical studies. This study explores whether metabolic signatures could be used as biomarkers for the multi-AGC kinase inhibitor AT13148. OBJECTIVES: To identify metabolomic changes of biomarkers of multi-AGC kinase inhibitor AT13148 in cells, xenograft / mouse models and in patients in a Phase I clinical study. METHODS: HILIC LC-MS/MS methods and Biocrates AbsoluteIDQ™ p180 kit were used for targeted metabolomics; followed by multivariate data analysis in SIMCA and statistical analysis in Graphpad. Metaboanalyst and String were used for network analysis. RESULTS: BT474 and PC3 cells treated with AT13148 affected metabolites which are in a gene protein metabolite network associated with Nitric oxide synthases (NOS). In mice bearing the human tumour xenografts BT474 and PC3, AT13148 treatment did not produce a common robust tumour specific metabolite change. However, AT13148 treatment of non-tumour bearing mice revealed 45 metabolites that were different from non-treated mice. These changes were also observed in patients at doses where biomarker modulation was observed. Further network analysis of these metabolites indicated enrichment for genes associated with the NOS pathway. The impact of AT13148 on the metabolite changes and the involvement of NOS-AT13148- Asymmetric dimethylarginine (ADMA) interaction were consistent with hypotension observed in patients in higher dose cohorts (160-300 mg). CONCLUSION: AT13148 affects metabolites associated with NOS in cells, mice and patients which is consistent with the clinical dose-limiting hypotension.

Subunit arrangement and phenylethanolamine binding in GluN1/GluN2B NMDA receptors.

Since it was discovered that the anti-hypertensive agent ifenprodil has neuroprotective activity through its effects on NMDA (N-methyl-D-aspartate) receptors, a determined effort has been made to understand the mechanism of action and to develop improved therapeutic compounds on the basis of this knowledge. Neurotransmission mediated by NMDA receptors is essential for basic brain development and function. These receptors form heteromeric ion channels and become activated after concurrent binding of glycine and glutamate to the GluN1 and GluN2 subunits, respectively. A functional hallmark of NMDA receptors is that their ion-channel activity is allosterically regulated by binding of small compounds to the amino-terminal domain (ATD) in a subtype-specific manner. Ifenprodil and related phenylethanolamine compounds, which specifically inhibit GluN1 and GluN2B NMDA receptors, have been intensely studied for their potential use in the treatment of various neurological disorders and diseases, including depression, Alzheimer's disease and Parkinson's disease. Despite considerable enthusiasm, mechanisms underlying the recognition of phenylethanolamines and ATD-mediated allosteric inhibition remain limited owing to a lack of structural information. Here we report that the GluN1 and GluN2B ATDs form a heterodimer and that phenylethanolamine binds at the interface between GluN1 and GluN2B, rather than within the GluN2B cleft. The crystal structure of the heterodimer formed between the GluN1b ATD from Xenopus laevis and the GluN2B ATD from Rattus norvegicus shows a highly distinct pattern of subunit arrangement that is different from the arrangements observed in homodimeric non-NMDA receptors and reveals the molecular determinants for phenylethanolamine binding. Restriction of domain movement in the bi-lobed structure of the GluN2B ATD, by engineering of an inter-subunit disulphide bond, markedly decreases sensitivity to ifenprodil, indicating that conformational freedom in the GluN2B ATD is essential for ifenprodil-mediated allosteric inhibition of NMDA receptors. These findings pave the way for improving the design of subtype-specific compounds with therapeutic value for neurological disorders and diseases.

Targeting triple-negative breast cancer through the somatostatin receptor with the new cytotoxic somatostatin analogue AN-162 [AEZS-124].

Previously, we have shown that the targeted cytotoxic somatostatin (sst) analogue AN-162 [AZSE-124] inhibits the growth of MDA-MB-231 human breast cancers xenografted into nude mice. In this study, we examined the trafficking of AN-162 into the cell, the expression of the somatostatin receptors (sstr) in specimens of human triple-negative breast cancers (TNBC), and the effect of AN-162 on HCC 1806 human TNBC xenografts. The expression of sstr in TNBC tumor samples was investigated by immunohistochemical staining. The expression of sstr in HCC 1806 was evaluated by reverse transcription PCR. Internalization studies with I-labeled AN-162 were carried out and the autofluorescence sign of doxorubicin moiety in the cell nucleus after incubation with AN-162 was measured using a fluorescence assay. The effects of AN-162 on the growth of HCC 1806 xenografted into nude mice were studied. A fluorescence microscopy cytotoxicity assay in vitro to detect cell death after treatment with AN-162 was also carried out. About 28% of TNBC tumor specimens showed a positive staining for sstr subtype 2a. HCC 1806 expresses all five subtypes of sstr. In the fluorescence cytotoxicity assay, dead HCC 1806 cells were found 24 h after incubation with AN-162. The growth of HCC 1806 tumors in nude mice was significantly inhibited by treatment with AN-162. AN-162 was internalized into the HCC 1806 cells and doxorubicin moiety was detected in the cell nuclei. This study is the first to show that the trafficking of the cytotoxic sst analogue AN-162 into the cell is mediated by sstr. Our work shows that the growth of xenografted HCC 1806 TNBCs can be effectively inhibited in vivo with AN-162. This investigation provides information on the mechanism of action and efficacy of this new targeted cytotoxic sst analogue and identifies in this relation the sstr as a favorable therapeutic target in TNBC.

Expression of neuropeptide hormone receptors in human adrenal tumors and cell lines: antiproliferative effects of peptide analogues.

Peptide analogues targeting various neuropeptide receptors have been used effectively in cancer therapy. A hallmark of adrenocortical tumor formation is the aberrant expression of peptide receptors relating to uncontrolled cell proliferation and hormone overproduction. Our microarray results have also demonstrated a differential expression of neuropeptide hormone receptors in tumor subtypes of human pheochromocytoma. In light of these findings, we performed a comprehensive analysis of relevant receptors in both human adrenomedullary and adrenocortical tumors and tested the antiproliferative effects of peptide analogues targeting these receptors. Specifically, we examined the receptor expression of somatostatin-type-2 receptor, growth hormone-releasing hormone (GHRH) receptor or GHRH receptor splice variant-1 (SV-1) and luteinizing hormone-releasing hormone (LHRH) receptor at the mRNA and protein levels in normal human adrenal tissues, adrenocortical and adrenomedullary tumors, and cell lines. Cytotoxic derivatives of somatostatin AN-238 and, to a lesser extent, AN-162, reduced cell numbers of uninduced and NGF-induced adrenomedullary pheochromocytoma cells and adrenocortical cancer cells. Both the splice variant of GHRH receptor SV-1 and the LHRH receptor were also expressed in adrenocortical cancer cell lines but not in the pheochromocytoma cell line. The GHRH receptor antagonist MZ-4-71 and LHRH antagonist Cetrorelix both significantly reduced cell growth in the adrenocortical cancer cell line. In conclusion, the expression of receptors for somatostatin, GHRH, and LHRH in the normal human adrenal and in adrenal tumors, combined with the growth-inhibitory effects of the antitumor peptide analogues, may make possible improved treatment approaches to adrenal tumors.

The inhibitory effect of a novel cytotoxic somatostatin analogue AN-162 on experimental glioblastoma.

Glioblastoma multiforme is the most common and most aggressive type of high grade tumor with a poor prognosis upon discovery. Based on earlier promising results earned with AN-162, a doxorubicin molecule linked to somatostatin (SST) analogue RC-160, it was our aim to determine the effect of AN-162 on DBTRG-05 glioblastoma cell line, and to test its efficacy in experimental brain tumors. We detected the expression of mRNA for somatostatin receptor (SSTR) subtypes 2 and 3 in DBTRG-05 cells with RT-PCR. Using ligand competition assay, specific high affinity receptors for somatostatin were found. The MTT assay showed that both AN-162 and doxorubicin (DOX) significantly inhibited cell proliferation and that there was no significant difference between the effects in vitro. Nude mice were xenografted with DBTRG-05 glioblastoma tumors. AN-162 showed a significant inhibition of tumor growth compared with the control group and the groups treated with equimolar doses of doxorubicin, somatostatin analogue RC-160, or the unconjugated mixture of doxorubicin plus RC-160. The tumor doubling time in the group of animals treated with AN-162 was extended and was significantly different from doubling times in the control group and in the other treatment groups. Our study clearly demonstrates a potent inhibitory effect of AN-162 in experimental glioblastoma, thus suggesting the possibility of its utilization in patients suffering from malignant brain cancer.

Preclinical evaluation of properties of a new targeted cytotoxic somatostatin analog, AN-162 (AEZS-124), and its effects on tumor growth inhibition.

In view of findings that various tumors express receptors for somatostatin, a new targeted cytotoxic analog of somatostatin, AN-162 (AEZS-124), consisting of doxorubicin linked through glutaric acid to the somatostatin octapeptide RC-121 was developed in our laboratory. We studied the toxicity in vivo and the effect of AN-162 on growth of the MDA-MB-231 estrogen-independent human breast cancer cell line xenografted into nude mice. AN-162 induced significant tumor growth inhibition compared with the control and the group treated with doxorubicin in equimolar doses. We also evaluated the stability of AN-162 in various sera in vitro, as this conjugate is susceptible to hydrolysis by serum carboxylesterase enzymes in the circulation. This study shows for the first time that AN-162 is a safe and effective compound for the treatment of experimental breast cancer. Our findings support the concept of targeted chemotherapy based on cytotoxic peptide analog AN-162 for the treatment of breast cancers and other cancers expressing somatostatin receptors.

Rho Kinase Inhibition by AT13148 Blocks Pancreatic Ductal Adenocarcinoma Invasion and Tumor Growth.

The high mortality of pancreatic cancer demands that new therapeutic avenues be developed. The orally available small-molecule inhibitor AT13148 potently inhibits ROCK1 and ROCK2 kinases that regulate the actomyosin cytoskeleton. We previously reported that ROCK kinase expression increases with human and mouse pancreatic cancer progression and that conditional ROCK activation accelerates mortality in a genetically modified LSL-KrasG12D; LSL-p53R172H; Pdx1-Cre; (KPC) mouse pancreatic cancer model. In this study, we show that treatment of KPC mouse and human TKCC5 patient-derived pancreatic tumor cells with AT13148, as well as the ROCK-selective inhibitors Y27632 and H1152, act comparably in blocking ROCK substrate phosphorylation. AT13148, Y27632, and H1152 induced morphologic changes and reduced cellular contractile force generation, motility on pliable discontinuous substrates, and three-dimensional collagen matrix invasion. AT13148 treatment reduced subcutaneous tumor growth and blocked invasion of healthy pancreatic tissue by KPC tumor cells in vivo without affecting proliferation, suggesting a role for local tissue invasion as a contributor to primary tumor growth. These results suggest that AT13148 has antitumor properties that may be beneficial in combination therapies or in the adjuvant setting to reduce pancreatic cancer cell invasion and slow primary tumor growth. AT13148 might also have the additional benefit of enabling tumor resection by maintaining separation between tumor and healthy tissue boundaries.Significance: Preclinical evaluation of a small-molecule ROCK inhibitor reveals significant effects on PDAC invasion and tumor growth, further validating ROCK kinases as viable therapeutic targets in pancreatic cancer. Cancer Res; 78(12); 3321-36. ©2018 AACR.

Role of the anti-glioma drug AT13148 in the inhibition of Notch signaling pathway.

OBJECTIVE: To investigate the drug targets related to Notch signaling pathway for glioma treatment. METHODS: Gene expression profiles GSE44561, GSE48079 and GSE22772GSE48079GSE22772 of glioma cells samples with activated Notch signaling pathway and control samples were downloaded from Gene Expression Omnibus database to screen the differentially expressed genes (DEGs) using limma package. GO (Gene Oncology) function and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analyses were conducted using DAVID tools to predict the underlying function of these DEGs. Sequentially, drug target genes recorded in DrugBank database were collected and matched with the selected DEGs to identify the potential drug targets for glioma. Further, these targets were verified by the screened DEGs in the anti-glioma drug (AT13148) treated samples of microarray data of GSE38008. RESULTS: A total of 75,645,497 DEGs were respectively identified in GSE44561, GSE48079 and GSE22772GSE48079GSE22772 datasets and these DEGs could well distinguish the glioma samples from controls. The DEGs were mainly enriched in classical functions and pathways, such as cell cycle, and DNA replication. A total of 122 DEGs were found to be potential drug targets for glioma, among which GLIPR1 was targeted by drug XL820, PDGFRB and KDR were targeted by SOT-107. Efficacy validation of the other 119 drug targets by GSE38008 data showed that ACSS1, ASL, GCLM, ROCK2, IMPA1, and TFPI may be targeted by the anti-glioma drug of AT13148. CONCLUSION: AT13148 may inhibit glioma progression by suppressing the Notch signaling genes, including GLIPR1, PDGFRB, ACSS1, and ASL.

Hemodynamic effects of intravenous butopamine in congestive heart failure.

Butopamine is chemically similar to dobutamine but, unlike dobutamine, it is not a catecholamine. Preclinical studies on dogs show that butopamine is inotropic intravenously and orally. We gave butopamine intravenously to eight patients with congestive heart failure using a progressive dose-response protocol ranging from 0.02 to 0.17 mcg/kg/min. The mean cardiac index and stroke volume index increased at doses greater than or equal to 0.06 mcg/kg/min; there was also an increase in heart rate at greater than or equal to 0.06 mcg/kg/min. At greater than or equal to 0.08 mcg/kg/min the augmented stroke volume tended to plateau so that additional increases in the cardiac index were secondary to the elevated heart rate. Improved ventricular performance, measured by systolic time intervals, left ventricular stroke work index, and the calculated mean rate of left ventricular pressure development during isovolumetric contraction (delta P/delta t/PCWP), was noted at greater than or equal to 0.06 mcg/kg/min. Systemic systolic blood pressure increased at greater than or equal to 0.04 mcg/kg/min but diastolic and mean arterial pressures and pulmonary artery and pulmonary capillary wedge pressures did not change. The progressive increase in cardiac output was accompanied by a reduction in pulmonary and systemic vascular resistances. Although mean premature ventricular contractions per minute did not change, two patients experienced a substantial increase in ventricular ectopy at 0.10 and 0.12 mcg/kg/min. Butopamine induces a positive inotropic response in patients with congestive heart failure but for equal increments in cardiac output, butopamine increases heart rate more than dobutamine.

Limited potentiation of blood pressure response to oral tyramine by brain-selective monoamine oxidase A-B inhibitor, TV-3326 in conscious rabbits.

TV-3326 is a novel cholinesterase inhibitor that produces irreversible brain-selective inhibition of monoamine oxidase (MAO)-A and B and has antidepressant-like activity in rats after chronic oral administration. This study determined whether TV-3326 would cause less potentiation than other irreversible MAO-inhibitors of the blood pressure (BP) response to oral tyramine in conscious rabbits. Dose-response curves were established for the increase in BP induced by tyramine (5-200 mg/kg) administered orally via a naso-pharyngeal tube. From these, the dose that increased BP by 30 mmHg (ED(30)) was computed for each rabbit before and after oral administration of clorgyline, 1 mg/kg for one week, tranylcypromine 10 mg/kg, once, moclobemide, 20 mg/kg 3 times and TV-3326, 26 mg/kg for 2 weeks. Clorgyline, tranylcypromine and TV-3326 inhibited brain MAO-A by 90%; the former two inhibited intestinal MAO-A by 85-97% but TV-3326 had no effect. Tranylcypromine and clorgyline produced 6 and 20-fold increases in the pressor response to tyramine while TV-3326, like moclobemide, only potentiated it 2-fold. If TV-3326 is found to produce as little potentiation of the tyramine response in human subjects, it may be a potentially useful therapeutic agent for the treatment of Alzheimer's disease with depression.

Importance of the aromatic ring in adrenergic amines. 6. Nonaromatic analogues of phenylethanolamine as inhibitors of phenylethanolamine N-methyltransferase: role of pi-electronic and steric interactions.

To probe the importance of pi-electronic and steric interactions of nonaromatic analogues of phenylethanolamine as inhibitors of phenylethanolamine N-methyltransferase (PNMT), a series of norbornane and norbornene ethanolamines was prepared and evaluated as inhibitors of PNMT (liquid chromatographic-electrochemical detector assay). Previous studies indicated a major importance of hydrophobic interaction of the ring moiety attached to the ethanolamine side chain, but a possible importance of pi-complex formation could have been obscured by conformational differences among the analogues. In this study, norbornane and norbornene substituted with an ethanolamine side chain at positions 1,2-exo, and 2-endo were prepared from the corresponding aldehydes by addition of trimethylsilyl cyanide (Me3SiCN) and lithium aluminum hydride reduction. The saturated (norbornane) analogues were two times more potent as inhibitors of the enzyme than were the norbornene analogues, thus suggesting that pi-complex formation is not an important contribution to binding and, as previously proposed, a hydrophobic interaction is the significant binding interaction of the ring moiety. The hydrophobic binding area has a critical size that requires the hydrophobic moiety to be of sufficient length (the bridgehead-substituted norbornane and norbornene ethanolamines being too "'short" for optimal binding). The 2-exo orientation of the ethanolamine side chain was preferred to the 2-endo orientation, supporting our earlier hypothesis that the ring moiety prefers to be oriented away from the side chain.

Activities of octopamine and synephrine stereoisomers on alpha-adrenoceptors.

1. The activities of the (-)- and (+)-forms of m- and p-octopamine and m- and p-synephrine on alpha 1-adrenoceptors from rat aorta and anococcygeus and alpha 2-adrenoceptors from rabbit saphenous vein were compared with those of noradrenaline (NA). 2. The rank order of potency of the (-)-forms on alpha 1-adrenoceptors from rat aorta and alpha 2-adrenoceptors was NA greater than m-octopamine = m-synephrine greater than p-octopamine = p-synephrine. The two m-compounds were 6 fold less active than NA on alpha 1-adrenoceptors from rat aorta and 150 fold less active on alpha 2-adrenoceptors. The two p- compounds were 1,000 fold less active than NA on both alpha 1-adrenoceptors from rat aorta and alpha 2-adrenoceptors. The rank order of potency of the (-)- forms on alpha 1-adrenoceptors from rat anococcygeus was NA = m-synephrine greater than m-octopamine greater than p-octopamine = p-synephrine. m-Octopamine was 4 fold less active than NA and (-)-m-synephrine. The two p- compounds were 30 fold less active than NA. 3. The rank order of potency of the (+)- forms was NA greater than m-octopamine greater than m-synephrine greater than p-octopamine greater than p-synephrine on both alpha 1- and alpha 2-adrenoceptors. The potency of each (+)- form was 1-2 orders of magnitude less than that of the (-) counterpart, the differences being greater for the stereoisomers of synephrine than for those of octopamine on both alpha 1- and alpha 2-adrenoceptors. 4. The yohimbine diastereoisomer antagonists, rauwolscine and corynanthine, were tested against (-)-NA and (-)-m-octopamine-induced contractions in both preparations. Based upon the known selectivities of these isomers for alpha-adrenoceptor subtypes, it is concluded that the rat aorta contains only alpha 1-adrenoceptors while the rabbit saphenous vein possesses predominantly alpha 2-adrenoceptors. 5. Ligand binding data for the octopamine and synephrine stereoisomers at alpha 1- and alpha 2-binding sites from rat cerebral cortex was also obtained. (-)-Forms were more active than (+)-forms. The rank order of affinity of the (-)-forms for both alpha 1- and alpha 2-binding sites was NA greater than m-octopamine = m-synephrine greater than p-synephrine greater than p-octopamine. The relative affinities of the members of the series against alpha 1-binding sites were very similar to their relative functional activities on rat aorta. However, the affinities of both m- and p-compounds relative to that of ( -)-NA were much greater at the x2-binding sites than were the relative activities in rabbit saphenous vein, possibly suggesting low intrinsic efficacy. Functional antagonist responses to NA by the (-)-octopamine and synephrines could not, however, be demonstrated on rat aorta or rabbit saphenous vein. 6. The activities of m-octopamine and m-synephrine were not significantly different from each other on either a,-adrenoceptors from rat aorta or x2-adrenoceptors; however, m-synephrine is more active than m-octopamine on a,-adrenoceptors from rat anococcygeus. Both m-octopamine and msynephrine can be considered to be naturally occurring x,-selective amines. However, if m- and poctopamine are co-released with NA in amounts proportional to their concentration, it is concluded that their activities on m,- and x2-adrenoceptors are too low to be physiologically significant.

Effects of alpha 2-adrenergic agonists on carbachol-stimulated catecholamine synthesis in cultured bovine adrenal medullary cells.

We examined the effects of alpha 2- and alpha 1-adrenergic agonists on synthesis of catecholamines in cultured bovine adrenal medullary cells. Clonidine, an alpha 2-adrenergic agonist, inhibited carbachol-stimulated synthesis of [14C]catecholamines from [14C]tyrosine in a concentration-dependent manner. Clonidine also inhibited carbachol-induced uptake of 45Ca2+ into cells at concentrations similar to those that inhibited the synthesis of [14C]catecholamines. Other alpha 2-adrenergic agonists, oxymetazoline and guanfacine, also strongly inhibited carbachol-stimulated synthesis of [14C]catecholamines. alpha 1-Adrenergic agonists, phenylephrine and norfenefrine, did not affect the synthesis. Tyrosine hydroxylase (EC 1.14.16.2) activity in a soluble fraction of cultured bovine adrenal medullary cells was assayed after gel filtration on a Sephadex G-25 column. Stimulation of the cells with carbachol increased the activity of tyrosine hydroxylase. Clonidine, oxymetazoline, and guanfacine all suppressed the carbachol-induced increase in activity of tyrosine hydroxylase in the cells. These results suggest that alpha 2-adrenergic agonists inhibit carbachol-stimulated synthesis of catecholamines by suppression of tyrosine hydroxylase activity, probably through the inhibition of Ca2+ uptake. However, the involvement of alpha 2-adrenoceptors in the inhibitory effects of alpha 2-agonists on catecholamine synthesis is still unsettled, since yohimbine failed to antagonize the inhibitory effect of clonidine on the synthesis in cultured bovine adrenal medullary cells.

Effects of catecholamines, monophenolamines and phenylamines on identifiable giant neurons of an African giant snail (Achatina fulica Férussac).

The effects of catecholamines, monophenolamines and phenylamines on eight identifiable giant neurons of an African giant snail (Achatina fulica Férussac) were examined to classify these neurons into several categories according to their sensitivities to the various substances. The five neurons, PON (periodically oscillating neuron), TAN (tonically autoactive neuron), 1-VMN (left-visceral multiple spike neuron), d-RPeAN (dorsal-right pedal autoactive neuron) and VIN (visceral intermittent firing neuron), were sensitive to catecholamines. Of these neurons, PON was excited most markedly by dopamine (dopamine-sensitive); TAN, 1-VMN and d-RPeAN were inhibited most markedly by epinine (epinine-sensitive); and VIN was excited equally by the four catecholamines, dopamine, epinine, L-noradrenaline and L-adrenaline (widely sensitive). The three other neurons, FAN (frequently autoactive neuron), d-LPeLN (dorsal-left pedal large neuron) and d-LCDN (dorsal-left cerebral distinct neuron) were sensitive to monophenolamines. DL-Octopamine was the most inhibitory on FAN and d-LCDN, but was the most excitatory on d-LPeLN. DL-Synephrine had the same but somewhat weaker effects on the three neurons as did DL-octopamine. The three phenylamines, L-phenylalanine, beta-phenylethylamine and DL-beta-phenylethanolamine, had no effect on any of the eight neurons examined.

The effects of beta-phenylethylamine and phenylethanolamine on water intake in rats: a temporal analysis.

Male Wistar rats were adapted to a restricted access schedule of water intake. For each animal overall water intake (ml) and number of licks per minute were measured over the period of access to water. The effects of various doses of beta-phenylethylamine and of phenylethanolamine were assessed in separate groups of animals. In terms of effects on overall consumption the two amines were equipotent in suppressing water intake. The local analysis revealed, however, that beta-phenylethylamine was more potent, but had a much shorter duration of action than phenylethanolamine.

MAO-B inhibitor deprenyl and beta-phenylethylamine potentiate [D-ALA2]-Met-enkephalinamide-induced seizures.

The relationship between deprenyl (MAO-B inhibitor), beta-phenylethylamine (PEA, MAO-B substrate) and [D-Ala2]-Met-enkephalinamide (DALA)-induced seizure was studied in the urethane-anaesthetized rats. A combined electromyographic (EMG) and electrocorticographic (ECoG) method was used. PEA (20-100 micrograms ivt) or DALA (10 micrograms ivt) induced myoclonic contractions (MC) in the submandibular muscle and epileptiform pattern with spike activity in the ECoG. Administration of subconvulsant doses of PEA (5-10 micrograms ivt 0.5-1 min before DALA) significantly increased DALA-induced seizure activity. Similarly, blockade of MAO-B with deprenyl (3-48 mg/kg ip) also enhanced DALA-induced epileptiform pattern. It is evident from this study that MAO-B system significantly modulates the excitatory phenomena induced by DALA. These findings of interactions between MAO-B system and enkephalinergic one, might be of relevance in the clinical situations such as psychosis, stress, a use of tricyclic antidepressants and all other cases, where the alteration of MAO-B system is a part of disease or induced during drug therapy.

Effect of intracerebroventricularly administered octopamines and synephrines on angiotensin II-induced water intake in rats.

Recent studies from this laboratory showed that l-m-synephrine (phenylephrine), a metabolite of l-m-octapamine, inhibited the drinking response of rats to peripherally administered angiotensin II. The objective of this investigation was to determine whether the isomers of both octapamine and synephrine could inhibit angiotensin II-induced dipsogenesis in the rat. Of the isomers tested, only d,l-m-octopamine and l-m-synephrine blocked the dipsogenic response to administration of angiotensin II (200 micrograms/kg, SC). The antidipsogenic effect of both d,l-m-octopamine and l-m-synephrine could be blocked by concurrent administration of yohimbine (300 micrograms/kg, IP), an alpha 2-adrenoceptor antagonist. The results indicate that m-octopamine and m-synephrine exert their antidipsogenic effect via alpha 2-adrenoceptors. These studies add to a growing body of data suggesting that activation of alpha 2-adrenoceptors inhibits, while blockade of these receptors enhances, angiotensin II-induced drinking.

Stimulation of cyclic AMP and lipolysis in adipose tissue of normal and obese Avy/a mice by LY79771, a phenethanolamine, and stereoisomers.

The stimulation of cyclic AMP and lipolysis by LY79771, a phenethanolamine antiobesity compound, and its 3 stereoisomers in adipose tissue of obese viable yellow mice and normal mice were studied. Both activities were stereo-specific with LY79771, the R,S isomer, and LY79730, the R,R isomer, being more potent than LY103085, the S,S isomer, and LY103672, the S,R isomer. Propranolol, a nonspecific beta-antagonist, completely inhibited the elevation of cyclic AMP and lipolysis whereas atenolol, a specific beta 1 antagonist, inhibited the elevation of cyclic AMP but did not completely inhibit lipolysis. These findings indicate that the elevation of cyclic AMP was mediated by the beta 1-receptor whereas the stimulation of lipolysis was mediated by both the beta 1 and beta 2 receptors. The adipose tissue of the obese viable yellow mice responded to these compounds less than that of the normal mice.

Myogenic effect of SP-1f and SP-1h two novel ß3-adrenoceptor (ß3-AR) agonists in human colonic circular smooth muscle.

The effect of two novel ß3-adrenoceptor (ß3-AR) agonists SP-1f and SP-1h on human colon circular smooth muscle contractility and ß3-AR mRNA expression have been determined. ß3-AR is ascertained co-participates to the control of the gut motility. Isometric tension on human colon muscle strips was measured in response to increasing concentrations of SP-1f, SP-1h and (-)-isoprenaline, alone and in the presence of Betaxolol, ICI 11,855 and SR 59230A (ß1-, ß2- and ß3-AR antagonists, respectively). (-)-Isoprenaline concentration-dependently relaxed circular muscle strips with an EC50=0.32±0.06µM. Such an effect was antagonized either by the contemporaneously presence of Betaxolol and ICI 11,855 [(-)-isoprenaline EC50=1.75±0.35µM, pKB=7.88±0.10] or by Betaxolol, ICI 11,855 and SR 59230A [(-)-isoprenaline EC50=3.49±0.38µM, pKB=8.51±0.14]. Besides, SP-1f and SP-1h concentration-dependently relaxed circular muscle strips with an EC50=0.35±0.07µM and 0.45±0.12µM, respectively. These values remained unchanged by blocking the ß1- and ß2-AR. The presence of SR 59230A antagonized the relaxing effect of SP-1f (EC50=3.51±0.94µM, pKB=8.93±0.16) and did not modify the SP-1h relaxing potency. In colon circular smooth muscle and in mucosa, ß3-AR mRNA expression levels were found to be 0.39±0.70 and 0.26±0.12 (P<0.05), respectively. Such results provide further evidence of the ß3-adrenoceptor functional role in the human colon and the crucial contribution of SP-1f to the control of the gut dysmotility.

Anti-tumor effects of peptide analogs targeting neuropeptide hormone receptors on mouse pheochromocytoma cells.

Pheochromocytoma is a rare but potentially lethal chromaffin cell tumor with currently no effective treatment. Peptide hormone receptors are frequently overexpressed on endocrine tumor cells and can be specifically targeted by various anti-tumor peptide analogs. The present study carried out on mouse pheochromocytoma cells (MPCs) and a more aggressive mouse tumor tissue-derived (MTT) cell line revealed that these cells are characterized by pronounced expression of the somatostatin receptor 2 (sst2), growth hormone-releasing hormone (GHRH) receptor and the luteinizing hormone-releasing hormone (LHRH) receptor. We further demonstrated significant anti-tumor effects mediated by cytotoxic somatostatin analogs, AN-162 and AN-238, by LHRH antagonist, Cetrorelix, by the cytotoxic LHRH analog, AN-152, and by recently developed GHRH antagonist, MIA-602, on MPC and for AN-152 and MIA-602 on MTT cells. Studies of novel anti-tumor compounds on these mouse cell lines serve as an important basis for mouse models of metastatic pheochromocytoma, which we are currently establishing.