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Purity: ≥98%
RO1138452 is a novel, potent and selective antagonist of the IP (prostacyclin) receptor. RO1138452 exhibits a strong IP receptor affinity. pKi is 9.3±0.1 in human platelets and 8.7±0.06 in a recombinant IP receptor system. RO1138452 has the ability to reduce pain and reduce inflammation. Significant reductions in acetic acid-induced abdominal constrictions are observed with RO1138452 (1-10 mg/kg, i.v.). RO1138452 (p.o., 3–100 mg/kg) dramatically lessens the mechanical hyperalgesia and edema formation caused by carrageenan. The total plasma concentration is 0.189 μg/mL one hour after rats were given RO1138452 (5 mg/kg, i.v.), while the free plasma concentration is calculated to be 0.009 μg/mL (28 nM).
Targets |
Rat I2 Receptor ( IC50 = 7 nM ); Rat I2 Receptor ( pKi = 8.33 ); Rabbit PAF Receptor ( IC50 = 52.9 nM );
Human α2A adrenoceptor ( IC50 = 724 nM ); Rat α1B adrenoceptor ( IC50 = 3280 nM ); Human Muscarinic M4 Receptor ( IC50 = 1450 nM ); Human muscarinic M2 Receptor ( IC50 = 2220 nM ); Human muscarinic M1 Receptor ( IC50 = 2570 nM ); Human muscarinic M5 Receptor ( IC50 = 3110 nM ); Rat 5-HT1B Receptor ( IC50 = 1130 nM ); pig 5-HT2C Receptor ( IC50 = 1190 nM ); Rat 5-HT2A Receptor ( IC50 = 3040 nM ); Human 5-HT1A Receptor ( IC50 = 8580 nM ); Guinea-pig 5-HT4 Receptor ( IC50 = 8910 nM ); Rat α2Badrenoceptor ( pKi = 5.87 ); Human α2A adrenoceptor ( pKi = 6.49 ); Human muscarinic M1 Receptor ( pKi = 5.66 ); Human muscarinic M5 Receptor ( pKi = 5.81 ); Human muscarinic M2 Receptor ( pKi = 5.88 ); Human muscarinic M4 Receptor ( pKi = 6.14 ); Rabbit PAF Receptor ( pKi = 7.9 ); Guinea-pig 5-HT4 Receptor ( pKi = 5.35 ); Human 5-HT1A Receptor ( pKi = 5.37 ); Rat 5-HT2A Receptor ( pKi = 5.71 ); Rat 5-HT1B Receptor ( pKi = 6.11 ); Pig 5-HT2C Receptor ( pKi = 6.11 ) |
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ln Vitro |
RO1138452 (CAY10441) is IP receptor antagonist. RO1138452 has a pIC50 value of 7.0±0.07 for attenuating cAMP accumulation. By quantifying the inhibition of carbaprostacyclin-induced cAMP accumulation in CHO-K1 cells that are stable in expressing the human IP receptor, the functional antagonistic effects of RO1138452 are investigated. RO1138452's antagonist affinity (pKi) is 9.0±0.06. A panel of receptor binding and enzyme tests is used to determine the selectivity profiles for RO1138452. At imidazoline2 (I2) (8.3) and platelet activating factor (PAF) (7.9) receptors, RO1138452 exhibits affinity[1]. When added to cells in conjunction with a fixed concentration of Taprostene (1 μM), RO1138452 (10 pM–10 μM) inhibits the inhibition of CXCL9 and CXCL10 release in a concentration-dependent manner, with p[A]50 (molar) values of -8.73±0.11 and -8.47±0.16 (p>0.05), respectively[2].
Affinity estimates of RO1138452 (CAY10441) and RO3244794 for human IP receptors [1] We estimated the binding affinity (pKi) of RO1138452 and RO3244794 in two independent systems using competition displacement assays from 4 to 5 independent experiments: human platelet membranes (Figure 2a) and CHO-K1 membranes expressing the recombinant human IP receptor (Figure 2b). For the native and the recombinant human IP receptor systems, the affinity estimates of iloprost were 8.3±0.2 and 8.4±0.1, respectively. RO1138452 was more potent than RO3244794 in displacing 3H-labelled iloprost in both systems studied. In human platelets, affinities of RO1138452 and RO3244794 were 9.3±0.1 and 7.7±0.03, respectively. In the recombinant IP receptor system, the affinities of RO1138452 and RO3244794 were estimated to be 8.7±0.1 and 6.9±0.1, respectively. In both the platelet and the recombinant systems, the Hill coefficients did not differ significantly from unity. Functional antagonism of cPGI2-induced cAMP accumulation [1] In order to determine whether RO1138452 (CAY10441) and RO3244794 behave as functional antagonists of the IP receptor, we tested whether these two compounds could block cAMP accumulation in CHO-K1 cells overexpressing the human IP receptor in 4–5 independent experiments (Figure 3). cPGI2 was used as the agonist to stimulate the IP receptor in these cells and showed a potency (pEC50) of 10±0.08. For cAMP inhibition experiments, 10 nM cPGI2 was used to drive the cAMP signalling pathway after incubating cells with various concentrations of RO1138452 and RO3244794. Consistent with the binding affinities, RO1138452 was a more potent antagonist of the human IP receptor than RO3244794. The pIC50 values of RO1138452 and RO3244794 in attenuating cAMP accumulation were 7.0±0.07 and 6.5±0.06, respectively. Using the Cheng–Prusoff correction factor (Cheng & Prusoff, 1973), functional estimates of affinities for RO1138452 and RO3244794 were 9.0±0.06 and 8.5±0.11, respectively. The reversibility of both compounds remains to be characterized. Neither RO1138452 nor RO3304794 showed any agonist activity at the recombinant IP receptor (data not shown). Neither compounds inhibited platelet aggregation, independent of stimulus (ADP or adrenaline), as would be expected of an agonist at the native IP receptor (data not shown). Selectivity of RO1138452 (CAY10441) or RO3244794 [1] Receptor selectivity profiles for RO1138452 (CAY10441) and RO3244794 were determined by evaluating the effects of the IP receptor ligands on specific binding of standard radioligands to more than 50 receptors. Full displacement curves were constructed with those receptor subtypes at which RO1138452 and RO3244794 (10 μM) inhibited specific radioligand binding by >70 and >30%, respectively, in an initial screen: α1B and α2A adrenoceptor, I2, muscarinic (M1–M5), platelet activating factor (PAF) and 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2C and 5-HT4. Potency (IC50) and affinity (pKi) estimates of RO1138452 and reference standards at these receptor subtypes are indicated in Table 1. Except for the I2 and PAF receptor, RO1138452 displayed relatively low affinities at all other receptor subtypes tested. RO1138452 did not exhibit any significant potency at non-IP prostanoid receptors (EP1, EP3, FP and TP; data not shown). As a result of intrinsically lower IP receptor affinity, the threshold for displacement by RO3244794 (10 μM) at the receptor subtypes in the selectivity panel was lowered to 30%. In this experiment, RO3244794 displaced 30% or more of the radioligands for the following receptor subtypes: adenosine (A3), EP1, EP3, EP4 and TP. RO3244794 displayed relatively weak affinities at these four receptor subtypes (Table 1), with no activity at I2 or PAF receptors. We also determined RO1138452 (CAY10441) and RO3244794 to be weak inhibitors of COX-1 and COX-2 enzymes. RO1138452 (CAY10441) (10 μM) produced less than 10% inhibition of ram COX-1, but inhibited the activity of human COX-2 by 29%. Likewise RO3244794 (10 μM) did not inhibit human COX-1 activity, but produced comparable inhibition of human COX-2 (27%). The extent to which the prostacyclin (IP) receptor regulates the release of two proinflammatory chemokines from human airway epithelial cells was investigated using the novel and competitive IP-receptor antagonist 4,5-dihydro-1H-imidazol-2-yl)-[4-(4-isopropoxy-benzyl)-phenyl]-amine (RO1138452 (CAY10441)). In BEAS-2B human airway epithelial cells, taprostene, a selective IP-receptor agonist, suppressed interferon-gamma-induced CXCL9 and CXCL10 release in a concentration-dependent manner. These effects were mimicked by 8-bromo-cAMP, and they were abolished in cells infected with an adenovirus vector encoding a highly selective inhibitor of cAMP-dependent protein kinase (PKA). RO1138452 blocked the inhibitory effect of taprostene on chemokine output in a manner inconsistent with surmountable competitive antagonism. Comparable results were obtained using primary cultures of human airway epithelial cells. The basis of the antagonism imposed by RO1138452 was studied further using BEAS-2B cells stably transfected with a cAMP-response element (CRE) luciferase reporter. On this output, RO1138452 also behaved insurmountably. Mechanistically, this could not be attributed to covalent receptor inactivation, allosterism, or a state of hemiequilibrium. Other studies established that the degree by which RO1138452 (CAY10441) antagonized taprostene-induced CRE-dependent transcription was not reversed over a 20-h "washout" period. This pharmacological profile is consistent with the behavior of a pseudo-irreversible antagonist where dissociation from its cognate receptor is so slow that re-equilibration is not achieved at the time the response is measured. Collectively, these data provide compelling evidence that human airway epithelial cells express inhibitory IP-receptors linked to the activation of PKA. Moreover, contrary to existing literature, RO1138452 behaved pseudoirreversibly, emphasizing the need, in drug discovery, to screen potential new medicines in the target tissue(s) of interest [2]. |
ln Vivo |
RO1138452 (CAY10441) has the ability to act as an analgesic and an anti-inflammatory and is a strong and selective antagonist for both rat and human IP receptors. Significant reductions in acetic acid-induced abdominal constrictions are observed with RO1138452 (1-10 mg/kg, i.v.). RO1138452 (p.o., 3–100 mg/kg) dramatically lessens the mechanical hyperalgesia and edema formation caused by carrageenan. Rats were given RO1138452 (5 mg/kg, i.v.) for one hour, during which time the total plasma concentration was 0.189 μg/mL and the free plasma concentration was calculated to be 0.009 μg/mL (28 nM)[1].
Tests of nociception, hyperalgesia and inflammation [1] Intraperitoneal injection of an irritant, such as acetic acid, provokes prostanoid-dependent chemical nociception characterized by abdominal constrictions (Berkenkopf & Weichman, 1988). To determine if IP receptors mediate the response, the two selective IP receptor antagonists were evaluated for their effects on acetic acid-induced abdominal constrictions in rats. In vehicle-treated rats, acetic acid induced 12±1.7 constrictions during the 15-min observation period (Figure 5a). RO1138452 (CAY10441) (1–10 mg kg−1, i.v., n=6–8) significantly (P<0.05) inhibited constrictions by 65±10% at 10 mg kg−1, i.v. (see Figure 5a). In a separate study (Figure 5b), acetic acid induced 9.8±0.8 constrictions and RO3244794 (1–30 mg kg−1, i.v., n=8) significantly (P<0.01) and dose-dependently decreased the number of constrictions by 100% at the highest dose level, with an ED50 value of 3.8±1.03 mg kg−1. Injection of carrageenan into the rat hind paw elicits a persistent inflammatory response characterized, in part, by mechanical hyperalgesia (Vinegar et al., 1976). To determine the involvement of the IP receptor in carrageenan-induced mechanical hyperalgesia, the effects of selective IP receptor antagonists administered 2 h after carrageenan treatment and 1 h before testing were assessed. In vehicle-treated, naïve rats (not treated with carrageenan), the paw withdrawal threshold was 125±6 g, whereas the withdrawal threshold in rats pretreated with vehicle and carrageenan was 55±5 g with the lower threshold reflecting mechanical hyperalgesia (Figure 6a). RO1138452 (CAY10441) (3–100 mg kg−1, p.o., n=10) significantly (P<0.01) increased the withdrawal threshold in a dose-dependent manner to 113±5 g, (84±8% inhibition of hyperalgesia) with an ED50 value of 18.3±1.9 mg kg−1 (Figure 6a). In a separate study (Figure 6b), in which carrageenan pretreatment reduced the paw withdrawal threshold from 220±15 g in naïve control rats to 104.5±4.9 g, RO3244794 (1–30 mg kg−1, p.o., n=10) significantly (P<0.01) increased the paw withdrawal threshold in a dose-dependent manner to a maximum of 192±13.5 g (76±12% inhibition of hyperalgesia), with an ED50 value of 14±3.6 mg kg−1. This response did not result from sedation, since a higher dose (100 mg kg−1, p.o.) does not affect locomotor activity in rats (data not shown). Injection of carrageenan into the rat hind paw also induces the formation of edema, which, in part, is attenuated by COX enzyme inhibitors (Vinegar et al., 1976). To determine the role of the IP receptor in prostanoid-dependent component of edema formation, the effects of the two selective IP receptor antagonists were evaluated on carrageenan-induced edema, with the compounds being administered immediately before injection of carrageenan. In vehicle treated rats, carrageenan stimulated 0.71±0.04 ml (Figure 7a) of edema. Indomethacin (10 mg kg−1, p.o.) significantly (P<0.01) reduced the edema to 0.37±0.03 ml, a 100% reduction of the prostanoid-dependent edema formation. Under the same conditions, RO1138452 (CAY10441) (3–100 mg kg−1, p.o. n=10) significantly (P<0.01) decreased edema to 0.48±0.03 ml, a 77±27% reduction of prostanoid-dependent edema at the highest dose level tested (Figure 7a). In a separate study (Figure 7b), carrageenan-stimulated edema in vehicle treated rats was 0.56±0.03 g. Indomethacin (5 mg kg−1, p.o.) significantly (P<0.01) reduced the edema to 0.32±0.04 g. RO3244794 (0.3–10 mg kg−1, p.o., n=10) significantly (P<0.01) decreased edema formation in a dose-dependent manner to 0.33±0.03 g, a 99±7% reduction of prostanoid-dependent edema. This value was not significantly different from that produced by indomethacin. A higher dose (30 mg kg−1) did not further reduce edema formation (data not shown). Intra-articular injection of mIOA produces a chronic osteoarthritic-like condition in rats, presumably associated with joint discomfort (Bove et al., 2003). Since RO3244794 showed superior pharmacokinetics with generally better in vivo potency and efficacy as compared with RO1138452 (CAY10441), it was further characterized in the mIOA model of osteoarthritis in rats. RO3244794 (1 and 10 mg kg−1, p.o., n=10) significantly (P<0.05) reduced the difference in weight distribution between the osteoarthritic and control hind paws, reflecting amelioration of joint discomfort (Figure 8). At 1 mg kg−1, the difference in weight distribution was reduced from 24.8±4.4 to 12.8±4.2 g (P<0.05) at 1 h following dosing compared with vehicle, which increased the weight distribution slightly from 31.2±7.8 to 33.1±5.7 g during the same 1 h. At 10 mg kg−1, the difference in weight distribution decreased from 22.1±5.4 to 0.34±7.5 g (P<0.01) at 1 h following dosing, compared with the vehicle control; this constituted a 99±23% reduction. Activity seen with both doses of RO3244794 at 1 h was sustained to at least 3 h. Further, the effects achieved with both doses of RO3244794 were similar in magnitude to that obtained with the selective COX-2 inhibitor, rofecoxib (10 mg kg−1), the positive control. |
Enzyme Assay |
RO1138452 (CAY10441)'s (10 μM) capacity to displace particular standard radioligand binding at 51 receptors is what determines its selectivity. For RO1138452, complete concentration-dependent displacement curves are constructed in triplicate and used to generate IC50 values when a significant displacement of radioligand is observed (>70%). The EP3 receptor is subjected to displacement binding. There are also experiments with enzyme inhibition. COX isoforms, namely COX-1 (ram seminal vesicle) and COX-2 (sheep placenta and human umbilical vein), are assessed for inhibition using RO1138452 at 10 μM in triplicate. The substrate utilized is arachidonic acid, and PGE2 accumulation is observed[1].
Native IP receptor in human platelets [1] Human platelets were first centrifuged at low speed (∼800 × g) for 5 min on a table top centrifuge. The supernatant was then centrifuged at 17,000 r.p.m. (45,400 × g) for 30 min at 4°C. The pellet was resuspended in membrane buffer (20 mM Tris-HCl, 5 mM EDTA), homogenized using a polytron homogenizer (setting 5) and centrifuged again at 45,400 × g for 30 min at 4°C. The pellet was then re-suspended in 20 mM Tris-HCl, 5 mM MgCl2, homogenized, aliquoted and stored at −80°C until used. For competition displacement binding experiments, increasing concentrations of iloprost, RO1138452 (CAY10441) and RO3244794 were used to compete with 10 nM 3H-labelled iloprost binding. Briefly, 50 μg of platelet membrane was used for each reaction and nonspecific binding was determined in the presence of 10 μM unlabelled iloprost, while total binding was determined in the presence of 8.5 nM 3H-labelled iloprost. The incubation time was 90 min at 25°C and the reaction was terminated by vacuum filtration using Whatman GF/B filtration plates. Scintillant was then added and the radioactivity counted using a TopCount microplate scintillation counter. Functional assay (cAMP) of IP receptor antagonism [1] IP receptor-transfected CHO-K1 cells were cultured in Ham's F-12 nutrient media supplemented with 10% foetal bovine serum and G418 (300 μg ml−1), harvested at 90% confluence, washed twice with PBS and detached with VERSENE for 5 min at 37°C. Cells were then re-suspended in 40 ml of stimulation buffer (Hank's buffered salt solution with 5 mM HEPES, 0.1% bovine serum albumin) and centrifuged at 800 × g for 5 min. After centrifugation, the pellet was suspended in stimulation buffer (with 0.5 M isobutylmethyl xanthine, IBMX). Cells were diluted to the appropriate number of cells ml−1 for a plating density of 100,000 cells well−1. cAMP detection was carried out using the AlphaScreen™ assay platform in a 96-well format. For inhibition experiments, 5 μl of either RO1138452 (CAY10441) or RO3244794 (in stimulation buffer) was dispensed to a 96-well plate in triplicate. Cell suspensions (10 μl) were added with anti-cAMP acceptor beads in stimulation buffer to each plate and incubated for 15 min at room temperature (in the dark or covered with a black plate). Then 5 μl of agonist or vehicle was added to each well; for the wells containing antagonist, 10 nM of carbaprostacyclin (cPGI2), a stable PGI2 analogue, was used. Plates were incubated for 30 min at room temperature (in the dark or covered with a black plate) before the addition of 10 μl donor beads with biotin-cAMP in lyses buffer (5 mM HEPES, 0.3% Tween-20, 0.1% bovine serum albumin). Plates were incubated for 1 h with gentle shaking (in the dark or covered with a black plate). Plates were read on an AlphaScreen™ Fusion analyzer. Receptor/enzyme profiling [1] Selectivity was determined by the ability of RO1138452 (CAY10441) or RO3244794 (10 μM) to displace specific binding of standard radioligands at 51 receptors. When significant displacement of radioligand was observed (>70% for RO1138452 (CAY10441) and >30% for RO3244794), complete concentration-dependent displacement curves (in triplicate) were constructed to generate IC50 values. Displacement binding at the EP3 receptor was performed at Roche Palo Alto. Enzyme inhibition assays were also conducted by Cerep according to standardized protocols. RO1138452 was evaluated at 10 μM in triplicate for inhibition of COX isoforms:COX-1 (ram seminal vesicle), COX-2 (sheep placenta and human umbilical vein). Arachidonic acid was used as a substrate and PGE2 accumulation was detected by Flash Plate™. For RO3244794 (10 μM), human COX-2 activity was determined using arachidonic acid as a substrate (with PMA as COX-2 inducer) and PGE2 accumulation was determined by radioimmunoassay. |
Cell Assay |
BEAS-2B cells are cultured in supplement-free keratinocyte serum-free medium (KSFM) with and without 100 nM RO1138452 for 30 minutes at 37°C. Following a wash in supplement-free KSFM, the cells are exposed to 1 μM Taprostene and incubated in the same medium for predetermined lengths of time. After harvesting the cells in reporter lysis buffer for four hours, luciferase activity is determined. By measuring the amount that mitochondrial dehydrogenases convert the tetrazolium salt MTT to formazan, colorimetric analysis is used to assess the viability of HAECs and BEAS-2B cells[2].
Recombinant IP receptor in Chinese hamster ovary (CHO)-K1 cells [1] For displacement of 3H-labelled iloprost with RO1138452 (CAY10441), membranes were precoupled to wheat germ agglutinin-coupled scintillation proximity assay beads (WGA-SPA beads by suspending the beads in assay buffer at 500 mg/25 ml. Equal volumes of suspended beads and resuspended membranes were mixed and placed on an orbital shaker maintained at 300 r.p.m. for 2 h. The receptor precoupled beads were then centrifuged at 300–500 × g for 7 min and the pellet was washed once with assay buffer. The final pellet was brought up to the original volume with assay buffer. For displacement of 3H-labelled iloprost by cold iloprost and RO3244794, regular filtration methodology was used as described for the native human platelets. Increasing concentrations of iloprost, RO1138452 (CAY10441) and RO3244794 were used to displace 7.5 nM (for WGA-SPA assay) or 12 nM (for filtration assay) 3H-labelled iloprost. |
Animal Protocol |
Rats: RO1138452 (CAY10441) (5 mg/kg, i.v.) is given to three male Sprague-Dawley rats. The rats are given a dose and then given 5% halothane to induce anesthesia. Blood is drawn from the rats' orbits using an orbital bleed into a syringe that has been heparinized. The blood is centrifuged at 2600× g for 5 minutes in a clinical centrifuge to extract the plasma fraction. Using high-performance liquid chromatography and mass spectrometry for detection, the amount of RO1138452 in each sample is ascertained[1].
RO1138452 (CAY10441) (2–10 ml kg−1, <10 mg ml−1) was readily dissolved in saline or water. RO3244794 (1 ml kg−1, <30 mg ml−1) was dissolved in one of several vehicles: (a) 100 mM Trizma® base; (b) 10% DMSO, 50% propylene glycol in deionized water and (c) 5.6% sodium benzoate, 0.5% benzoic acid, 85% propylene glycol or suspended in 0.5% carboxymethylcellulose, 0.9% sodium chloride, 0.4% polysorbate, 0.9% benzyl alcohol in deionized water. [1] Abdominal constrictions test [1] Male Sprague–Dawley rats (∼120 g) were intravenously (i.v.) administered vehicle (1 ml kg−1), indomethacin (5–10 mg kg−1) or test compound (n=6–8). After 60 min, acetic acid (1%, 2 ml kg−1) in deionized water was injected into the peritoneum as described previously (Jett et al., 1999). The number of abdominal constrictions followed by dorsiflexion and extension occurring during a 15-min period beginning 15 min after acetic acid administration was counted. The data are expressed as mean (±s.e.m.) number of abdominal constrictions per 15 min period. Carrageenan-induced paw hyperalgesia test [1] Male Sprague–Dawley rats (∼120 g, n=10) were anesthetized with halothane (5%) and administered 100 μl of vehicle or carrageenan (1% in saline) subcutaneously (s.c.) on the plantar surface of the left hind paw, as described previously (Jett et al., 1999). Vehicle (1 ml kg−1) or test compound were administered orally (p.o.) 2 h after carrageenan administration and 1 h before evaluation of hind paw mechanical hyperalgesia, measured as the change in paw withdrawal threshold (g) at which a rat removes its hind paw, vocalizes or struggles, using the Ugo Basile Analgesy meter. Data are expressed as mean (±s.e.m.) paw withdrawal threshold (g). Carrageenan-induced edema formation test [1] Male Sprague–Dawley rats (∼130–140 g, n=10) were assigned to treatment groups so that each group was weight balanced and administered vehicle, indomethacin (positive control) or test compound. Immediately thereafter, the rats were anesthetized with halothane (5%) and administered 50 μl of vehicle or carrageenan (0.5% in saline) s.c. on plantar surface of the left hind paw, as described previously (Jett et al., 1999). After 3 h, the volume or weight of the treated and untreated hind paws was recorded and the difference between the paws was calculated. Both methods used to measure edema formation produced comparable results. Data are expressed as mean (±s.e.m.) difference in volume (ml) or weight (g), as a reflection of edema formation. Monoiodoacetate-induced osteoarthritis test [1] To induce symptoms of osteoarthritis, male Wistar rats (170–180 g, n=10) were anesthetized with isoflurane (2% in O2), administered a single intraarticular injection of sodium monoiodoacetate (mIOA; 1 mg in 50 μl) and returned to the animal colony (Bove et al., 2003). After 14 days, the rats were evaluated for joint discomfort, using an Incapacitance Tester, which measures the weight distribution between the right (injected) and left (control) hind paws, with the difference in weight distribution being an index of joint discomfort. The rats were tested before and after administration of vehicle, rofecoxib (positive control) or test compound. Data are expressed as mean (±s.e.m.) difference in weight distribution (g) between the osteoarthritic and control hind paws. Pharmacokinetic analysis [1] Male Sprague–Dawley rats (n=3) were administered RO1138452 (CAY10441) or RO3244794 (5 mg kg−1, i.v.). At various times after dose administration, the rats were anesthetized by halothane (5%), blood was collected by orbital bleed into a heparinized syringe and a plasma fraction was obtained by centrifugation of the blood at 2600 × g for 5 min in a clinical centrifuge. The level of test compound in each sample was determined by high-performance liquid chromatography with detection by mass spectrometry. Data are expressed as mean (±s.d. mean). Values for plasma half-lives, volume of distribution, etc., were calculated using WinNonlin. |
ADME/Pharmacokinetics |
Pharmacokinetics [1]
RO1138452 (CAY10441) and RO3244794 displayed dissimilar pharmacokinetic properties (Table 2). RO1138452 showed a shorter plasma half-life, lower plasma protein binding, a larger volume of distribution (Vdβ) and a lower total plasma concentration than RO3244794 following i.v. administration to rats. However, despite the difference in total plasma concentration following equivalent i.v. doses of the two compounds (Figure 4), the free plasma levels could be similar if the higher plasma protein binding of RO3244794 is taken into account. For example, 1 h after administration of RO1138452 and RO3244794 (5 mg kg−1, i.v.) to rats, the total plasma concentrations were 0.189 and 3.57 μg ml−1, respectively, whereas the free plasma concentrations were calculated to be 0.009 and 0.005 μg ml−1 (28 and 11 nM), respectively. RO3244794 also has greater oral bioavailability than RO1138452 in rats: 50.8 versus 0.69%, respectively. |
References |
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Additional Infomation |
Prostacyclin (PGI2) possesses various physiological functions, including modulation of nociception, inflammation and cardiovascular activity. Elucidation of these functions has been hampered by the absence of selective IP receptor antagonists. Two structurally distinct series of IP receptor antagonists have been developed: 4,5-dihydro-1H-imidazol-2-yl)-[4-(4-isopropoxy-benzyl)-phenyl]-amine (RO1138452) and R-3-(4-fluoro-phenyl)-2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-propionic acid (RO3244794).RO1138452 (CAY10441) and RO3244794 display high affinity for IP receptors. In human platelets, the receptor affinities (pKi) were 9.3 +/- 0.1 and 7.7 +/- 0.03, respectively; in a recombinant IP receptor system, pKi values were 8.7 +/- 0.06 and 6.9 +/- 0.1, respectively. Functional antagonism of RO1138452 and RO3244794 was studied by measuring inhibition of carbaprostacyclin-induced cAMP accumulation in CHO-K1 cells stably expressing the human IP receptor. The antagonist affinities (pKi) of RO1138452 and RO3244794 were 9.0 +/- 0.06 and 8.5 +/- 0.11, respectively. Selectivity profiles for RO1138452 and RO3244794 were determined via a panel of receptor binding and enzyme assays. RO1138452 displayed affinity at I2 (8.3) and PAF (7.9) receptors, while RO3244794 was highly selective for the IP receptor: pKi values for EP1 (< 5), EP3 (5.38), EP4 (5.74) and TP (5.09). RO1138452 (1-10 mg kg(-1), i.v.) and RO3244794 (1-30 mg kg(-1), i.v.) significantly reduced acetic acid-induced abdominal constrictions. RO1138452 (3-100 mg kg(-1), p.o.) and RO3244794 (0.3-30 mg kg(-1), p.o.) significantly reduced carrageenan-induced mechanical hyperalgesia and edema formation. RO3244794 (1 and 10 mg kg(-1), p.o.) also significantly reduced chronic joint discomfort induced by monoiodoacetate. These data suggest that RO1138452 and RO3244794 are potent and selective antagonists for both human and rat IP receptors and that they possess analgesic and anti-inflammatory potential. [1]
It should be noted that the slightly higher ED50 values reported for RO1138452 (CAY10441) relative to RO3244794 following oral or i.v. administration in our in vivo rat assays – despite the higher in vitro affinity of the former for the IP receptor – may be due to the inferior pharmacokinetic profile of RO1138452 in rat. In contrast to RO3244794, which has a longer (3.4 h) plasma half-life and higher oral bioavailability, RO1138452 has a plasma half-life of 1.3 h and very low oral bioavailability. In addition, the in vivo potency of RO3244794 itself may be somewhat reduced by higher levels of plasma protein binding. [1] In conclusion, RO1138452 (CAY10441) and RO3244794 are novel IP receptor antagonists that display a high affinity for human and rodent IP receptors. In vitro studies have demonstrated that these compounds are selective for the IP receptor over the EP and other members of the prostanoid receptor family, as well as over many other receptors and enzymes. Both compounds have undergone extensive in vivo pharmacological evaluation that shows the analgesic and anti-inflammatory potential of IP receptor antagonists. Give the relatively high receptor affinity and selectivity, these compounds should provide useful insights into the physiology of PGI2 and the role of IP receptors in cardio-protection, blood flow regulation, pain and inflammation. After this manuscript was submitted, the authors became aware of a publication in press (Nakae et al., 2005b) describing the biological activities of two compounds belonging to the same structural series as RO3244794 (Cournoyer et al., 2001). These data appear to provide confirmation of some of the in vitro findings reported in this paper. |
Molecular Formula |
C19H23N3O
|
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Molecular Weight |
309.4054
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Exact Mass |
309.184
|
Elemental Analysis |
C, 73.76; H, 7.49; N, 13.58; O, 5.17
|
CAS # |
221529-58-4
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PubChem CID |
9839644
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Appearance |
Light yellow to brown solid powder
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Density |
1.1±0.1 g/cm3
|
Boiling Point |
449.7±47.0 °C at 760 mmHg
|
Flash Point |
225.8±29.3 °C
|
Vapour Pressure |
0.0±1.1 mmHg at 25°C
|
Index of Refraction |
1.597
|
LogP |
3.17
|
Hydrogen Bond Donor Count |
2
|
Hydrogen Bond Acceptor Count |
2
|
Rotatable Bond Count |
6
|
Heavy Atom Count |
23
|
Complexity |
380
|
Defined Atom Stereocenter Count |
0
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SMILES |
O(C([H])(C([H])([H])[H])C([H])([H])[H])C1C([H])=C([H])C(=C([H])C=1[H])C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])N([H])C1=NC([H])([H])C([H])([H])N1[H]
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InChi Key |
GYYRMJMXXLJZAB-UHFFFAOYSA-N
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InChi Code |
InChI=1S/C19H23N3O/c1-14(2)23-18-9-5-16(6-10-18)13-15-3-7-17(8-4-15)22-19-20-11-12-21-19/h3-10,14H,11-13H2,1-2H3,(H2,20,21,22)
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Chemical Name |
N-[4-[(4-propan-2-yloxyphenyl)methyl]phenyl]-4,5-dihydro-1H-imidazol-2-amine
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Synonyms |
RO 1138452; CAY10441; RO-1138452; 221529-58-4; RO1138,452; Ro-1138,452; CAY10441; N-[4-[(4-propan-2-yloxyphenyl)methyl]phenyl]-4,5-dihydro-1H-imidazol-2-amine; CAY-10,441; N-(4-(4-isopropoxybenzyl)phenyl)-4,5-dihydro-1H-imidazol-2-amine; DH5W8F3S4H; RO1138452; CAY-10441; CAY 10441
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HS Tariff Code |
2934.99.9001
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Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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Solubility (In Vitro) |
DMSO: 62~100 mg/mL (200.4~323.2 mM)
Ethanol: ~50 mg/mL (~161.6 mM) |
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Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (8.08 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 2.5 mg/mL (8.08 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C,1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.  (Please use freshly prepared in vivo formulations for optimal results.) |
Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
1 mM | 3.2320 mL | 16.1598 mL | 32.3196 mL | |
5 mM | 0.6464 mL | 3.2320 mL | 6.4639 mL | |
10 mM | 0.3232 mL | 1.6160 mL | 3.2320 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration: mg/mL;
Method for preparing DMSO stock solution: mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation::Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.