Novel gonadotropin-releasing hormone antagonists with substitutions at position 5

Gonadotropin-releasing hormone (GnRH) antagonists with high potency and improved duration of action are needed for potential clinical applications. We synthesized four new antagonists (2-5) of GnRH homologues to Azaline B (1), with a common core sequence of [Aph(X)5, D-Aph(Cbm)6]Azaline B. In these analogs, (X) contains hydrophobic aromatic moieties (like homoveratoyl in 2, homovanillyl in 3, 2,5-dimethoxyphenylacetyl in 4, and 3,5-dimethoxyphenylacetyl in 5) designed to improve the duration of action over that of Azaline B. These analogs were tested in vitro for their ability to antagonize the GnRH receptor and in vivo for duration of action in a castrated male rat assay. Analogs 2, 4, and 5 were potent in vitro, but were found to be short acting in vivo. However, analog 3 [Aph(Hvn)5,D-Aph(Cbm)6]Azaline B is a potent human GnRH receptor antagonist in vitro (IC50 1.47 nM) and exhibits a longer duration of action than azaline B.     

Synthesis, in vivo and in vitro biological activity of novel azaline B analogs

Several azaline B analogs (2-10) were synthesized and evaluated for their ability to antagonize GnRH in vitro and for duration of action in inhibiting luteinizing hormone secretion in a castrated male rat assay in vivo. Analogs, 8 (IC(50) = 1.85 nM), and 9 (IC(50) = 1.78 nM), are equipotent with azaline B (1, IC(50) = 1.36 nM) in vitro. Whereas 9 is short acting, 8 is as long acting as azaline B. Other analogs have IC(50) greater than 2.0 nM and are all short acting.     

Short-chain pseudopeptide bombesin receptor antagonists with enhanced binding affinities for pancreatic acinar and Swiss 3T3 cells display strong antimitotic activity

The high inhibitory potency of the previously developed bombesin antagonist [Leu13, psi CH2NHLeu14]bombesin (analogue I) (IC50 values of 30 and 18 nM for inhibition of bombesin-stimulated amylase secretion from guinea pig acinar cells and Swiss 3T3 cell growth, respectively) diminished considerably when shorter chain lengths were examined. For instance, [Leu13, psi CH2NHLeu14]bombesin-(5-14),[Leu13, psi CH2NHLeu14] bombesin-(6-14), and [Leu9, psi CH2NHLeu10]neuromedin C had IC50 values of 150, 150, and 280 nM, respectively. Incorporation of a D-Phe residue at position 6 of [Leu13, psi CH2NHLeu14] bombesin did not significantly change the various biological parameters. However, its presence in [Leu13, psi CH2NHLeu14]bombesin-(6-14) and at position 2 of psi-neuromedin C-(2-10) resulted in about 10-fold increases in potency up to and above that of the original antagonist. For instance, [D-Phe6,Leu13,psi CH2NHLeu14]bombesin-(6-14) and des-Gly1-[D-Phe2,Leu9,psi CH2NHLeu10]neuromedin C exhibited IC50 values of 5 and 28 nM, respectively. Analogues based on the litorin sequence which contains an NH2-terminal pyroglutamic acid residue at the bombesin position 6 equivalent were also quite potent. The ability of various analogues to interact with bombesin receptors on pancreatic acini correlated reasonably well with potencies derived from inhibition of bombesin-stimulated growth of Swiss 3T3 cells. Additional studies of NH2- and COOH-terminal structure-activity relationships resulted in the synthesis of [D-Phe6,Leu13,psi CH2NHPhe14]bombesin-(6-14), which was particularly effective in inhibiting 3T3 cell growth at high picomolar concentrations (IC50 = 0.72 nM and Ki = 3.1 nM for 3T3 cells; IC50 = 7.5 nM and Ki = 9.9 nM for acini). Detailed investigations with one of the most potent antagonists, [D-Phe6,Leu13,psi CH2NHLeu14]bombesin-(6-14) (Ki = 14 nM for acini cells and 7.1 for 3T3 cells), demonstrated that this analogue was a competitive inhibitor of bombesin and that this activity was specific for the bombesin receptor. Thus, inhibitory potencies have been improved generally up to 25 times over previously reported structures; and, given that bombesin itself has a Ki of 1.2 nM for 3T3 cell binding, some of these analogues are extraordinarily high affinity receptor antagonists. They can also be synthesized more readily and offer fewer proteolytic degradation sites than the original pseudopeptide and should be excellent candidates for in vivo studies aimed at inhibition of bombesin-dependent human small cell lung carcinoma growth.     

Discovery of a novel CCR3 selective antagonist

In searching for a novel CCR3 receptor antagonist, we designed a library that included a variety of carboxamide derivatives based on the structure of our potent antagonists for human CCR1 and CCR3 receptors, and screened the new compounds for inhibitory activity against 125I-Eotaxin binding to human CCR3 receptors expressed in CHO cells. Among them, two 2-(benzothiazolethio)acetamide derivatives (1a and 2a) showed binding affinities with IC50 values of 750 and 1000 nM, respectively, for human CCR3 receptors. These compounds (1a and 2a) also possessed weak binding affinities for human CCR1 receptors. We selected la as a lead compound for derivatization to improve in vitro potency and selectivity for CCR3 over CCRI receptors. Derivatization of la by incorporating substituents into each benzene ring of the benzothiazole and piperidine side chain resulted in the discovery of a compound (1b) exhibiting 820-fold selectivity for CCR3 receptors (IC50 = 2.3 nM) over CCR1 receptors (IC50 = 1900 nM). This compound (1b) also showed potent functional antagonist activity for inhibiting Eotaxin (IC50 = 27 nM)- or RANTES (IC50 = 13 nM)-induced Ca2+ increases in eosinophils.     

Novel endomorphin-2 analogs with mu-opioid receptor antagonist activity.

A series of position 4-substituted endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) analogs containing 3-(1-naphthyl)-alanine (1-Nal) or 3-(2-naphthyl)-alanine (2-Nal) in L- or D-configuration, was synthesized. The opioid activity profiles of these peptides were determined in the mu-opioid receptor representative binding assay and in the Guinea-Pig Ileum assay/Mouse Vas Deferens assay (GPI/MVD) bioassays in vitro, as well as in the mouse hot-plate test of analgesia in vivo. In the binding assay the affinity of all new analogs for the mu-opioid receptor was reduced compared with endomorphin-2. The two most potent analogs were [D-1-Nal(4)]- and [D-2-Nal4]endomorphin-2, with IC50 values 14 +/- 1.25 and 19 +/- 2.1 nM, respectively, compared with 1.9 +/- 0.21 nM for endomorphin-2. In the GPI assay these analogs were found to be weak antagonists and they were inactive in the MVD assay. The in vitro GPI assay results were in agreement with those obtained in the in vivo hot-plate test. Antinociception induced by endomorphin-2 was reversed by concomitant intracerebroventricula (i.c.v.) administration of [D-1-Nal4]- and [D-2-Nal4]-endomorphin-2, indicating that these analogs were mu-opioid antagonists. Their antagonist activity was compared with that of naloxone. At a dose 5 microg per animal naloxone almost completely inhibited antinociceptive action of endomorphin-2, while [D-1-Nal4]endomorphin-2 in about 46%.     

R3(BDelta23 27)R/I5 chimeric peptide, a selective antagonist for GPCR135 and GPCR142 over relaxin receptor LGR7: in vitro and in vivo characterization

Both relaxin-3 and its receptor (GPCR135) are expressed predominantly in brain regions known to play important roles in processing sensory signals. Recent studies have shown that relaxin-3 is involved in the regulation of stress and feeding behaviors. The mechanisms underlying the involvement of relaxin-3/GPCR135 in the regulation of stress, feeding, and other potential functions remain to be studied. Because relaxin-3 also activates the relaxin receptor (LGR7), which is also expressed in the brain, selective GPCR135 agonists and antagonists are crucial to the study of the physiological functions of relaxin-3 and GPCR135 in vivo. Previously, we reported the creation of a selective GPCR135 agonist (a chimeric relaxin-3/INSL5 peptide designated R3/I5). In this report, we describe the creation of a high affinity antagonist for GPCR135 and GPCR142 over LGR7. This GPCR135 antagonist, R3(BDelta23-27)R/I5, consists of the relaxin-3 B-chain with a replacement of Gly23 to Arg, a truncation at the C terminus (Gly24-Trp27 deleted), and the A-chain of INSL5. In vitro pharmacological studies showed that R3(BDelta23-27)R/I5 binds to human GPCR135 (IC50=0.67 nM) and GPCR142 (IC50=2.29 nM) with high affinity and is a potent functional GPCR135 antagonist (pA2=9.15) but is not a human LGR7 ligand. Furthermore, R3(BDelta23-27)R/I5 had a similar binding profile at the rat GPCR135 receptor (IC50=0.25 nM, pA2=9.6) and lacked affinity for the rat LGR7 receptor. When administered to rats intracerebroventricularly, R3(BDelta23-27)R/I5 blocked food intake induced by the GPCR135 selective agonist R3/I5. Thus, R3(BDelta23-27)R/I5 should prove a useful tool for the further delineation of the functions of the relaxin-3/GPCR135 system.     

Real-time analysis of the activities of GnRH and GnRH analogs in alphaT3-1 cells by the Cytosensor microphysiometer

Gonadotropin-releasing hormone (GnRH), acting via the GnRH receptor, elicited rapid extracellular acidification responses in mouse gonadotrope-derived alphaT3-1 cells as measured by the Cytosensor microphysiometer, which indirectly monitors cellular metabolic rates. GnRH increased the extracellular acidification rate of the cells in a dose-dependent manner (EC(50) = 1.81 +/- 0.24 nM). The GnRH-stimulated acidification rate could be attenuated by protein kinase C (PKC) down-regulation, extracellular Ca2+ depletion, and the voltage-sensitive Ca2+ channel (VSCC) blocker nifedipine, indicating that the acidification response is activated by both Ca2+ and PKC-mediated pathways. Upon continuous exposure to 100 nM GnRH or periodic stimulation by 10 nM GnRH at 40 min intervals, homologous desensitization was more pronounced in the absence of extracellular Ca2+, suggesting that desensitization of GnRH activity may be mediated via depletion of intracellular Ca2+ stores. We have also compared the potency of eight GnRH analogs on alphaT3-1 cells. No acidification response was detected for GnRH free acid, consistent with the idea that the C-terminal amide is a critical structural determinant for GnRH activity. Replacement of Gly-NH(2) at the C-terminus by N-ethylamide dramatically reduced the EC(50) value, suggesting that substitution of the Gly-NH(2) moiety by N-ethylamide increases the potency of GnRH analogs. Substitution of Gly at position 6 by D-Trp significantly reduced the EC(50) value, whereas D-Lys at the same position slightly increased the EC(50) value, implying that either an aromatic amino acid or a non-basic amino acid at position 6 may be essential for potent GnRH agonists. In summary, our results demonstrate that the Cytosensor microphysiometer can be used to evaluate the actions of GnRH and GnRH analogs in alphaT3-1 cells in a real-time and noninvasive manner. This silicon-based microphysiometric system should provide new information on the structure-function studies of GnRH and is an invaluable tool for the screening of new GnRH agonists and antagonists in the future.     

Development of a phosphatase-resistant, L-tyrosine derived LPA1/LPA3 dual antagonist

Lysophosphatidic acid (LPA) is a bioactive compound that has gained attention due to its role in neoplastic diseases. Our group has developed a potent dual LPA1/LPA3 receptor antagonist, VPC51098 (LPA1 IC(50) = 84 nM, LPA1 IC(50) = 48 nM) that contained a labile phosphate head group. This lability has impaired our evaluation of our scaffold of LPA receptor antagonists in vivo. We wished to replace the phosphate with a potentially more stable head group while retaining potency at both LPA1 and LPA3 to facilitate future in vivo studies. We tested in vitro potency of all head groups including α-methylene, α-fluoromethylene, α-hydroxymethylene; vinyl phosphonates; α-fluoro vinyl phosphonates. The most potent compound was found to be a low micromolar inhibitor VPC51299 that contained a vinyl phosphonate and possessed a half-life of approximately 90 min in rats when dosed intravenously. Herein, we describe the synthesis and initial biological evaluation of these compounds.     

Synthesis, in vitro pharmacology, and structure-activity relationships of 2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid derivatives as mGluR2 antagonists

Chemical modification of the bicyclo[3.1.0]hexane ring C-3 position led to the discovery of 3-alkoxy-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid, 3-benzylthio-, and 3-benzylamino-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid derivatives, metabotropic glutamate receptor 2 (mGluR2) antagonists. In particular, 3-(3,4-dichlorobenzyloxy)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (15ae), (1R,2S,5R,6R)-2-amino-3-(3,4-dichlorobenzylthio)-6-fluorobicyclo[3.1.0]hexane-2,6-carboxylic acid (15at), and (1R,2S,5R,6R)-2-amino-3-(N-(3,4-dichlorobenzylamino))-6-fluorobicyclo[3.1.0]hexane-2,6-carboxylic (15ba) showed high affinity for the mGluR2 receptor (15ae: K(i) = 2.51 nM, 15at: K(i) = 1.96 nM, and 15ba: K(i) = 3.29 nM) and potent antagonist activity for mGluR2 (15ae; IC50 = 34.21 nM, 15at; IC50 = 13.34 nM, and 15ba; IC50 = 35.96 nM). No significant agonist activity for mGluR2 was observed with 15ae, 15at, or 15ba. This paper reports on the synthesis, in vitro pharmacological profile, and structure-activity relationships (SARs) of 3-substituted-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid.     

Arylacetamide kappa opioid receptor agonists with reduced cytochrome P450 2D6 inhibitory activity

Some kappa opioid receptor agonists of the arylacetamide class, for example, ICI 199441 (1), were found to strongly inhibit the activity of cytochrome P450 2D6 (CYP2D6) (1: CYP2D6 IC50=26 nM). Certain analogs bearing a substituted sulfonylamino group, for example, 13, were discovered to have significantly reduced CYP2D6 inhibitory activity (13: CYP2D6 IC50>10 microM) while displaying high affinity toward the cloned human kappa opioid receptor, good kappa/delta and kappa/mu selectivity, and potent in vitro and in vivo agonist activity.     

mCPP but not TFMPP is an antagonist at cardiac 5HT3 receptors.

The prototypic arylpiperazines, meta-chlorophenylpiperazine (mCPP), meta-trifluoromethylphenylpiperazine (TFMPP) and quipazine are widely studied serotonergic ligands with nonselective effects at 5HT1 and 5HT2 receptor subtypes. The present study was designed to compare the affinities of these arylipiperazines at 5HT3 receptors, and to determine agonist or antagonist activity at 5HT3 receptors. Quipazine showed high affinity at brain 5HT3 receptors (IC50 = 4.4 nM) and was a potent agonist of the von Bezold-Jarisch reflex in anesthetized rats, a response mediated by cardiac 5HT3 receptors. In concentrations that activated 5HT3 receptors, quipazine also antagonized serotonin-induced bradycardia in anesthetized rats. Taken together, these data suggest that quipazine is an agonist/antagonist with high affinity at 5HT3 receptors in both brain and cardiac tissue. Although mCPP also showed relatively high affinity at brain 5HT3 receptors (IC50 = 61.4 nM), it did not activate the von Bezold-Jarisch reflex; instead, mCPP potently antagonized serotonin-induced bradycardia. Thus, mCPP acts as an antagonist at 5HT3 receptors in the periphery. Although both quipazine and mCPP possessed relatively high affinity at brain 5HT3 receptors, TFMPP did not bind appreciably to 5HT3 receptors in brain (IC50 = 2373 nM) and neither activated nor inhibited cardiac 5HT3 receptors. That TFMPP did not interact with 5HT3 receptors, whereas quipazine and mCPP did, is in marked contrast to the similar effects of all three arylpiperazines at other serotonin receptors. The selectivity of TFMPP for 5HT1 and 5HT2 receptors (i.e., its minimal affinity for 5HT3 receptors) suggests that this arylpiperazine may be a preferred ligand relative to mCPP when studying 5HT1 or 5HT2 receptor mediated responses.     

Structure-function studies of linear and cyclized peptide antagonists of the GnRH receptor.

Structurally new analogs of the peptidic GnRH receptor antagonist Cetrorelix as well as conformationally constrained cyclized deca- or pentapeptides were synthesized and selected peptides evaluated comprehensively. To understand how structural variations of the antagonistic peptide effect pharmacodynamic properties, binding affinities and antagonistic potencies toward the human and rat GnRH receptor were determined. Whereas large substituents in position 6 of linear peptides are compatible with high binding affinity (K(D) < 0.5 nM), all cyclized peptides except the cyclo[3-10] analog D-52391 depicted low binding affinity (K(D) > 10 nM). Binding affinity and antagonistic potency in vitro correlated for all peptides and surprisingly no discrimination between human and rat receptor proteins was observed. Since receptor residues W(101) and N(102) are involved in agonist and antagonist binding, equally potent but structurally different antagonists were tested for binding to the respective W(101)A and N(102)A mutants. In contrast to linear decapeptides, residues N(102) and W(101) are not involved in binding of D-23938 and W(101) is the critical residue for D-52391 binding. We conclude that although equally potent, peptidic GnRH receptor antagonists do have distinct interactions within the ligand binding pocket. Finally, selected antagonists were tested for testosterone suppression in male rats. The duration of testosterone suppression below castration levels differed largely from 1 day for Ganirelix to 27 days for D-23487. Systemic availability became evident as the most important parameter for in vivo efficacy.     

Discovery of an orally bioavailable alkyl oxadiazole beta3 adrenergic receptor agonist

5-n-Pentyl oxadiazole substituted benzenesulfonamide 8 is a potent and selective beta3 adrenergic receptor agonist (beta3 EC50 = 23 nM, beta1 IC50 = 3000 nM, beta2 IC50 = 3000 nM). The compound has high oral bioavailability in dogs (62%) and rats (36%) and is among the most orally bioavailable beta3 adrenergic receptor agonists reported to date.     

In vitro and in vivo pharmacological characterization of SB 201993, an eicosanoid-like LTB4 receptor antagonist with anti-inflammatory activity.

Leukotriene B4 (LTB4) and 12-(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-[R]-HETE) have been postulated to contribute to the pathophysiology of inflammatory diseases. SB 201993, (E)-3-[[[[6-(2-carboxyethenyl)-5-[[8-(4-methoxyphenyl)octyl] oxy]-2-pyridinyl] methyl] thio] methyl] benzoic acid, identified from a chemical series designed as ring-fused analogs of LTB4, was evaluated as an antagonist of LTB4- and 12-(R)-HETE-induced responses in vitro and for anti-inflammatory activity in vivo. SB 201993 competitively antagonized [3-H]-LTB4 binding to intact human neutrophils (Ki = 7.6 nM) and to membranes of RBL 2H3 cells expressing the LTB4 receptor (RBL 2H3-LTB4R; IC50 = 154 nM). This compound demonstrated competitive antagonism of LTB4- and 12-(R)-HETE-induced Ca2+ mobilization responses in human neutrophils (IC50s of 131 nM and 105 nM, respectively) and inhibited LTB4-induced Ca2+ mobilization in human cultured keratinocytes (IC50 = 61 nM), RBL 2H3-LTB4R cells (IC50 = 255 nM) and mouse neutrophils (IC50 = 410 nM). SB 201993 showed weak LTD4-receptor binding affinity (Ki = 1.9 microM) and inhibited 5-lipoxygenase (IC50 of 3.6 microM), both in vitro and ex vivo. In vivo, SB 201993 inhibited LTB4-induced neutrophil infiltration in mouse skin and produced dose-related, long lasting topical anti-inflammatory activity against the fluid and cellular phases of arachidonic acid-induced mouse ear inflammation (ED50 of 580 microg/ear and 390 microg/ear, respectively). Similarly, anti-inflammatory activity was also observed in the murine phorbol ester-induced cutaneous inflammation model (ED50 of 770 and 730 microg/ear, respectively, against the fluid and cellular phases). These results indicate that SB 201993 blocks the actions of LTB4 and 12-(R)-HETE and inhibits a variety of inflammatory responses; and thus may be a useful compound to evaluate the role of these mediators in disease models.     

Synthesis and antihypertensive activity of pyrimidin-4(3H)-one derivatives as losartan analogue for new angiotensin II receptor type 1 (AT1) antagonists

The discovery, in vitro and in vivo studies of the highly potent AT(1) antagonist 12a (BR-A-657, Fimasartan) antagonists are presented. A series of pyrimidin-4(3H)-one derivatives as losartan analogue were synthesized and evaluated for a novel class of AT(1) receptor antagonists. Among them, 12a containing thioamido moiety displayed both high in vitro functional antagonism and binding affinity [IC(50)=0.42 and 0.13 nM, respectively] and inhibited strongly in vivo AngII-induced pressor response in pithed rats with an ED(50) of 0.018 mg/kg. Moreover, in vivo evaluation in furosemide-treated rat and conscious renal hypertensive rat models and the pharmacokinetic study showed that 12a is a highly potent and orally active AT(1) selective antagonist having stronger in vivo potency than losartan.     

Novel highly potent mu-opioid receptor antagonist based on endomorphin-2 structure

The mu-opioid agonists endomorphin-1 (Tyr-Pro-Trp-Phe-NH(2)) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2)) exhibit an extremely high selectivity for the mu-opioid receptor and thus represent a potential framework for modification into mu-antagonists. Here we report on the synthesis and biological evaluation of novel [d-2-Nal(4)]endomorphin-2 analogs, [Sar(2),d-2-Nal(4)]endomorphin-2 and [Dmt(1),Sar(2),d-2-Nal(4)]endomorphin-2 (Dmt=2'6'-dimethyltyrosine; Sar=N-methylglycine, sarcosine; d-2-Nal=3-(2-naphthyl)-d-alanine). [Dmt(1),Sar(2),d-2-Nal(4)]endomorphin-2 possessed very high affinity for the mu-opioid receptor (IC(50)=0.01+/-0.001 nM) and turned out to be a potent and extremely selective mu-opioid receptor antagonist, as judged by the in vitro aequorin luminescence-based calcium assay (pA(2)=9.19). However, in the in vivo hot plate test in mice this analog was less potent than our earlier mu-opioid receptor antagonist, [Dmt(1),d-2-Nal(4)]endomorphin-2 (antanal-2). The exceptional mu-opioid receptor in vitro activity and selectivity of [Dmt(1), Sar(2),d-2-Nal(4)]endomorphin-2 makes this analog a valuable pharmacological tool, but further modifications are needed to improve its in vivo profile.     

Synthesis and pharmacological evaluation of second-generation phosphatidic acid derivatives as lysophosphatidic acid receptor ligands

Short-chain phosphatidic acid derivatives, dioctanoyl glycerol pyrophosphate (DGPP 8:0, 1) and phosphatidic acid 8:0 (PA 8:0, 2), were previously identified as subtype-selective LPA(1) and LPA(3) receptor antagonists. Recently, we reported that the replacement of the phosphate headgroup by thiophosphate in a series of fatty alcohol phosphates (FAP) improves agonist as well as antagonist activities at LPA GPCR. Here, we report the synthesis of stereoisomers of PA 8:0 analogs and their biological evaluation at LPA GPCR, PPARgamma, and ATX. The results indicate that LPA receptors stereoselectively interact with glycerol backbone modified ligands. We observed entirely stereospecific responses by dioctyl PA 8:0 compounds, in which (R)-isomers were found to be agonists and (S)-isomers were antagonists of LPA GPCR. From this series, we identified compound 13b as the most potent LPA(3) receptor subtype-selective agonist (EC(50)=3 nM), and 8b as a potent and selective LPA(3) receptor antagonist (K(i)=5 nM) and inhibitor of ATX (IC(50)=600 nM). Serinediamide phosphate 19b was identified as an LPA(3) receptor specific antagonist with no effect on LPA(1), LPA(2), and PPARgamma.     

GIP-(3-42) does not antagonize insulinotropic effects of GIP at physiological concentrations

Glucose-dependent insulinotropic polypeptide [GIP-(1-42)] is degraded by dipeptidyl peptidase IV (DPP IV), forming GIP-(3-42). In mice, high concentrations of synthetic GIP-(3-42) may function as a GIP receptor antagonist, but it is unclear whether this occurs at physiological concentrations. In COS-7 cells transiently transfected with the human GIP receptor, GIP-(1-42) and -(3-42) bind with affinities (IC(50)) of 5.2 and 22 nM, respectively. GIP-(1-42) was a potent agonist, stimulating cAMP accumulation (EC(50), 13.5 pM); GIP-(3-42) alone had no effect. When incubated together with native GIP, GIP-(3-42) behaved as a weak antagonist (IC(50), 92 and 731 nM for inhibition of cAMP accumulation elicited by 10 pM and 1 nM native GIP, respectively). In the isolated perfused rat pancreas, GIP-(3-42) alone had no effect on insulin output and only reduced the response to GIP (1 nM) when coinfused in >50-fold molar excess (IC(50), 138 nM). The ability of GIP-(3-42) to affect the antihyperglycemic or insulinotropic actions of GIP-(1-42) was examined in chloralose-anesthetized pigs given intravenous glucose. Endogenous DPP IV activity was inhibited to reduce degradation of the infused GIP-(1-42), which was infused alone and together with GIP-(3-42), at rates sufficient to mimic postprandial concentrations of each peptide. Glucose, insulin, and glucagon responses were identical irrespective of whether GIP-(1-42) was infused alone or together with GIP-(3-42). We conclude that, although GIP-(3-42) can weakly antagonize cAMP accumulation and insulin output in vitro, it does not behave as a physiological antagonist in vivo.     

Identification of a dual δ OR antagonist/μ OR agonist as a potential therapeutic for diarrhea-predominant Irritable Bowel Syndrome (IBS-d)

A small set of acyclic analogs 5 were prepared to explore their structure-activity relationships (SARs) relative to heterocyclic core, opioid receptor (OR) agonists 4. Compound 5l was found to have very favorable OR binding affinities at the δ and μ ORs (r K(i) δ=1.3 nM; r K(i) μ=0.9 nM; h K(i) μ=1.7 nM), with less affinity for the κ OR (gp K(i) κ=55 nM). The OR functional profile for 5l varied from the previously described dual δ/μ OR agonists 4, with 5l being a potent, mixed dual δ OR antagonist/μ OR agonist [δ IC(50)=89 nM (HVD); μ EC(50)=1 nM (GPI); κ EC(50)=1.6 μM (GPC)]. Compound 5l has progressed through a clinical Phase II Proof of Concept study on 800 patients suffering from diarrhea-predominant Irritable Bowel Syndrome (IBS-d). This Phase II study was recently completed successfully, with 5l demonstrating statistically significant efficacy over placebo.     

Synthesis and biological evaluation of nucleobase-modified analogs of the anticancer compounds 3'-C-ethynyluridine (EUrd) and 3'-C-ethynylcytidine (ECyd)

A series of nucleobase-modified analogs of the anticancer compounds 3'-C-ethynyluridine (EUrd) and 3'-C-ethynylcytidine (ECyd) were designed to overcome the strict substrate specificity of the activating uridine-cytidine kinase. EUrd, ECyd and target nucleosides were obtained using a short convergent synthetic route utilizing diacetone-alpha-D-glucose as starting material. 5-Iodo-substituted EUrd was the most potent inhibitor among the novel nucleobase-modified analogs in in vitro assays against human adenocarcinoma breast and prostate cancer cells with IC50 values down to 35 nM.